Rapid and prolonged immunogic therapeutic

ABSTRACT

The present invention shows that intranasal administration of E1/E3-defective adenovirus particles may confer rapid and broad protection against viral and bacterial pathogens in a variety of disease settings. Protective responses lasted for many weeks in a single-dose regimen in animal models. When a pathogen-derived antigen gene was inserted into the E1/E3-defective adenovirus genome, the antigen-induced protective immunity against the specific pathogen was elicited before the adenovirus-mediated protective response declined away, thus conferring rapid, prolonged, and seamless protection against pathogens. In addition to E1/E3-defective adenovirus, other bioengineered non-replicating vectors encoding pathogen-derived antigens may also be developed into a new generation of rapid and prolonged immunologic-therapeutic (RAPIT).

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation in part of U.S. application Ser. No.13/426,037 filed Mar. 21, 2012, which claims priority to U.S.Provisional Patent Application No. 61/454,819 filed Mar. 21, 2011 and61/568,054 filed Dec. 8, 2011.

Reference is made to U.S. Pat. Nos. 6,348,450, 6,706,693, 6,716,823 and7,524,510, US Patent Publication Nos. 20030045492, 20030125278,20040009936, 20050271689, 20070178115, 20090175897 and 20110268762 andU.S. patent application Ser. No. 12/959,791.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

FEDERAL FUNDING LEGEND

This invention was supported, in part, by National Institutes of Healthgrants 2-R44-AI-068285-02, 1-UC1-AI-067198-01 and 1-UC1-A1-067205-1; aNational Institutes of Health contract N01-AI-30063; and a NationalInstitute of Allergy and Infectious Diseases Non-Clinical EvaluationAgreement. The federal government may have certain rights to thisinvention.

FIELD OF THE INVENTION

The present invention relates generally to the fields of immunology andtherapeutic technology. The present invention also relates to methods toelicit rapid-prolonged innate immune responses and uses thereof.

BACKGROUND OF THE INVENTION

The disease-fighting power of immunologics (e.g., vaccines) andtherapeutics (e.g., drugs) have been a public health bonanza creditedwith the worldwide reduction of mortality and morbidity. The goal tofurther amplify the power of medical intervention requires thedevelopment of a new generation of rapid-response immunologics that canbe mass produced at low costs and mass administered by nonmedicalpersonnel; as well as a new generation of therapeutics that can conferprolonged protection preferably not impaired by drug resistance. The newimmunologics and therapeutics also have to be endowed with a highersafety margin than that of conventional vaccines and drugs.

Use of conventional drugs against microbial pathogens often induces drugresistance over time because microbes constantly evolve under mutationalpressure. This invention illustrates that an anti-viral oranti-bacterial state can be rapidly induced in animals followingintranasal administration of an E1/E3-defective (ΔE1E3) adenovirusparticle by changing the habitat in the airway that impedes the growthof pathogens. Since the adenovirus particle does not directly attack apathogen, there is little chance for this novel therapeutic to inducedrug resistance. Furthermore, the adenovirus-induced anti-pathogen statecan persist for many weeks in animals, long enough for overlapping withthe induction of protective immunity elicited by a pathogen-derivedantigen expressed from the adenovirus, if a pathogen-derived antigen isinserted into the adenovirus genome as a vaccine. It is conceivable thata non-replicating adenovirus particle can be co-administered with othermucosal vaccines as a therapeutic adjunct.

The nonreplicating adenovirus-vectored vaccine holds promise in boostingvaccine coverage because the vector can be rapidly manufactured inserum-free suspension cells in response to a surge in demand. Moreover,preexisting adenovirus immunity does not interfere appreciably with thepotency of an adenovirus-vectored nasal vaccine. In addition to humanvaccination, animals can also be mass immunized by this class ofvectored vaccines.

There is a litany of demands for better vaccines. Although vaccinationproves to be the most cost-effective method for the prevention ofdisease, a sweeping offensive to boost vaccine coverage remains acompelling goal in the movement toward improved public health worldwide.Current vaccines that have been licensed for marketing include killedwhole microorganisms, live attenuated microorganisms, microbialextracts, purified or recombinant proteins, DNA vaccines and virus-likeparticles. Even though many diseases have been defeated by the broaddistribution of these vaccines, the goal to generate community (herd)immunity in a wide variety of disease settings remains elusive owing toa number of problems in current vaccination programs.

Specifically, vaccine-associated adverse side effects range from localand systemic inflammatory response, fever, platelet activation, cardiacautonomic dysfunction, anaphylactic reaction (induced by needleinjection of certain vaccines) [Salomon M E, Halperin R, Yee J.Evaluation of the two-needle strategy for reducing reactions to DPTvaccination. Am. J. Dis. Child. 141, 796-798 (1987), Lanza G A, BaroneL, Scalone G et al. Inflammation-relaxed effects of adjuvant influenza Avaccination on platelet activation and cardiac autonomic function. J.Intern. Med. 269, 118-125 (2011), Jae S Y, Heffernan K S, Park S H etal. Does an acute inflammatory response temporarily attenuateparasympathetic reactivation? Clin. Auton. Res. 20, 229-233 (2010) andSever J L, Brenner A I, Gale A D et al. Safety of anthrax vaccine: anexpanded review and evaluation of adverse events reported to the VaccineAdverse Event Reporting System (VAERS). Pharmacoepidemiol. Drug Saf. 13,825-840 (2004)] to the rare occurrence of paralytic poliomyelitis(mediated by ingestion of the oral polio vaccine) [Minor P.Vaccine-derived poliovirus (VDPV): impact on poliomyelitis eradication.Vaccine 27, 2649-2652 (2009)]; myopericarditis (induced by inoculationof the Dryvax smallpox vaccine) [Poland. G A, Grabenstein J D, Neff J M.The US smallpox vaccination program: a review of a large modern erasmallpox vaccination implementation program. Vaccine 23, 2078-2081(2005)] and Bell's palsy (induced by a bacterial toxin nasal adjuvant)[Lewis D J, Huo Z, Barnett S et al. Transient facial nerve paralysispalsy) following intranasal delivery of a genetically detoxified mutantof Escherichia coli heat labile toxin. PLoS ONE 4, e6999 (2009) andCouch R B. Nasal vaccination, Escherichia coli enterotoxin, and Bell'spalsy. N. Engl. J. Med. 350, 860-861 (2004)].

In 2010, a sudden rise of narcolepsy among vaccinees was reported in afew countries following needle injection of an H1N1 pandemic influenzavaccine containing the squalene adjuvant. Injection of, qualene alonecan induce rheumatoid arthritis in animals [Carlson B C, Jansson A M,Larsson A, Bucht A, Lorentzen J C. The endogenous adjuvant squalene caninduce a chronic T-cell-mediated arthritis in rats. Am. J. Pathol. 156,2057-2065 (2000)]. As emerging evidence shows that chronic, low-gradeinflammation is associated with cardiovascular disease [Finch C E,Crimmins E M. Inflammatory exposure and historical changes in humanlife-spans. Science 305, 17361739 (2004)], obesity [Gregor M F,Hotamisligil G S. Inflammatory mechanisms in obesity. Anna. Rev.Immunol. 29, 415-445 (2011)], diabetes [Gregor M F, Hotamisligil. G S.Inflammatory mechanisms in obesity. Annu. Rev. Immunol. 29, 415-445(2011)], cancer [O'Callaghan I) S, O'Donnell D, O'Connell F, O'Byrne KJ. The role of inflammation in the pathogenesis of non-small cell lungcancer. J. Thorac. Oncol. 5, 2024-2036 (2010)] and neurological disorder[Witte M E, Geurts J J, de Vries H E, van der Valk P, van Horssen J.Mitochondrial dysfunction: a potential link between neuroinflammationand neurodegeneration? Mitochondrion 10, 411-418 (2010)],vaccine-induced inflammation now needs focused attention.

Whether an acute inflammatory reaction induced by injection of animmunostimulating vaccine-adjuvant complex [Salomon M E, Halperin R, YeeJ. Evaluation of the two-needle strategy for reducing reactions to DPTvaccination. Am. J. Dis. Child. 141, 796-798 (1987), Lanza G A, BaroneL, Scalone G et al. Inflammation-related effects of adjuvant influenza Avaccination on platelet activation and cardiac autonomic function. J.Intern, Med. 269, 118-125 (2011) and Jae S Y, Heffernan K S, Park S H etal. Does an acute inflammatory response temporarily attenuateparasympathetic reactivation? Clin. Auton. Res. 20, 229-233 (2010)]could evolve into a chronic, low-grade inflammation and trigger any ofthese ailments in a subset of vaccinees over time is of paramountimportance in public health; however, this potential hazard has not beenrigorously investigated. Since the concept of vaccine safety is evolvingfrom ‘protection against pathogen-induced diseases’ to ‘no possibilityof inducing adverse consequences’, any known extraneous agents, toxicityand residual virulence found in a vaccine would not be allowed, and anypossibility of inducing unknown side effects (e.g., inflammation invital organs) should be avoided.

Mucosal and systemic immune responses are elicited and regulated with aconsiderable degree of independence and most vaccines have beenadministered invasively by intramuscular injection, which induces goodsystemic immunity but often weak mucosal immunity that is crucial indefense against mucosal pathogens (e.g., influenza virus, Mycobacteriumtuberculosis and HIV) [Gallichan W S, Rosenthal K L. Long-livedcytotoxic T lymphocyte memory in mucosal tissues after mucosal hut notsystemic immunization, J. Exp. Med. 184, 1879-1890 (1996) and Saurer L,McCullough K C, Summerfield A. In vitro induction of mucosa-typedendritic cells by all-trans retinoic acid. J. Immunol. 179, 3504-3514(2007)]. Efficient induction of mucosal immunity usually employs nasalor oral vaccination owing to the unique ability of resident mucosaldendritic cells (DCs) to induce IgA switching and to imprintmucosa-specific homing receptors (e.g., CCR9 and α4β7 integrin) onlymphocytes [Saurer L, McCullough K C, Summerfield A. In vitro inductionof mucosa-type dendritic cells by all-trans retinoic acid. J. Immunol.179,3504-3514 (2007) and Molenaar R, Greuter M. van der Marel A P et al.Lymph node strornal cells support dendritic cell-induced gut-homing of Tcells. J. Immunol. 183, 6395-6402 (2009)1.

In addition to weak mucosal immunity induced by an injectable vaccine,the syringe needle as a vaccine administration device also poses seriousproblems through intentional or inadvertent unsterile re-use,needlestick injury, improper waste disposal, as well as limitedinjection service by licensed medical personnel during a crisis [Tang DC, Van Kampen K R. Toward the development of vectored vaccines incompliance with evolutionary medicine. Expert Rev. Vaccines 7(4),399-402 (2008)1 Public fear of pointed needles (aichmophobia) playsanother role in hindering vaccine coverage. Some people may thus preferthe odds of getting a disease versus the odds of inflicting pain,injury, or death by systemic vaccination. Since the objective ofvaccination programs is to reduce the overall probability of infectionby generating community (herd) immunity, the mission will be underminedby a hold-off on vaccination owing to public fear of risks. To date,enabling technologies for reversing negative perceptions by developing anew generation of rapid-response vaccines that are safe, efficacious,painless and economical are emerging on the horizon.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

The present invention is based upon the inventor's serendipitous findingthat a transgene-free ΔE1E3 adenovirus empty particle or an adenovirusvector encoding a pathogen-derived antigen could elicit arapid-prolonged-broad protective response against pathogens in a varietyof disease settings when intranasally administered.

Without being bound by limitation, Applicant hypothesizes thatadenovirus may be involved in activating specific arms of innateimmunity that impede growth of respiratory mucosal pathogens.

The present invention relaters to a method of inducing a response in apatient in need thereof which may comprise administering to the patientan adenovirus that is defective or deleted in its E1 and/or E3 regionsin an amount effective to induce the response. In an advantageousembodiment, the patient may be a mammal.

In one embodiment, the adenovirus does not contain and express atransgene.

In another embodiment, the adenovirus may contain and express a nucleicacid molecule encoding a gene product. In particular, the adenovirus maycomprise an exogenous or heterologous nucleic acid molecule encoding apathogen-derived gene product that elicits protective immunity. Theexogenous or heterologous nucleic acid molecule may encode an epitope ofinterest. In particular, the exogenous or heterologous nucleic acidmolecule may encode one or more influenza virus; respiratory syncytialvirus (RSV); Bacillus anthracia; or other pathogen-derived epitopes ofinterest and/or one or more influenza antigens.

In an advantageous embodiment, the adenovirus may be a human adenovirus,In another embodiment, the immune response may be elicited within 24hours. In another embodiment, the administration results in a protectiveresponse from about one day to about 47 days.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings.

FIG. 1 shows rapid protection of mice against influenza virus challengesby intranasal administration of adenovirus particles.

FIG. 2 shows rapid protection of mice against anthrax by intranasaladministration of adenovirus particles.

FIGS. 3A and 3B depict prophylactic therapy against lethal challenge byA/PR/8/34 (PR8) in mice. Prophylactic therapy was performed by i.n.administration of Ad5 particles shortly before PR8 challenge. AdE/in/−2and AdE*/in/−2, i.n. administration of AdE on day −2; AdE/in/+1, i.n.administration of AdE 1 day post-PR8 challenge; AdE/im/−2, i.m.injection of AdE on day −2; AdNC/in/−2 and AdNC*/in/−2, i.n,administration of AdNC.H1.1 on day −2; AdNC/im/−2, i.m. injection ofAdNC.H1.1 on day −2; untreated control, Balb/c mice without treatmentprior to PR8 challenge; all groups were inoculated with AdE or AdNC.H1.1at a dose of 1.7×610⁶ ifu except AdE*/in/−2 and AdNC*/in/−2 groups thatreceived a dose of 1.7×610⁶ ifu; all groups were challenged by i.n.instillation of 4×LD₅₀ of PR8 on day 0; body weights were recorded dailyfor 18 days post-challenge with 30% body weight loss taken as thedisease endpoint; numbers in parentheses represent the number of animalsin each group.

FIGS. 4A and 4B depict protection of mice by Ad5-mediated prophylactictherapy and vaccination against a higher dose of PR8 challenge.AdNC/in/−47, i,n. administration of AdNC.H1.1 on day −47; AdE/in/−47,i.n. administration of AdE on day −47; AdE/in/−47-2, i.n. administrationof AdE on day −47 followed by a booster application of day −2;AdE/in/−1, i,n. administration of AdE on day −1; wtAd/in/−1, i.n.administration of E1+/E3+ wild-type Ad5 particles on day −1; all groupswere inoculated with Ad5 particles at a dose of 1.2×10⁸ ifu followed bychallenge with 10×LD₅₀ of PR8 on day 0; body weights were recorded dailyfor 14 days post-challenge; other symbols and protocols are the same asthose described in FIG. 3 legend.

FIG. 5 depicts health status of PR8-challenged animals as shown by bodyweight loss. Post-challenge body weights are presented as mean % bodyweight by taking the body weight of individual mice on day 1 as 100%.Symbols and challenge protocols are the same as those described in FIGS.3 and 4 legends. Although AdE/in/−47-2 and ADNC/in/−47 animals lost lessweight than mice in other groups, the difference did not reachstatistical significance (by one-way ANOVA with Turkey's multiplecomparison post-tests; the untreated control group was excluded instatistical analysis due to early termination of data points).

FIGS. 6A-H depicts Lung histopathology induced by PR8 infection. (A andE) Lung resected from an untreated control mouse (FIG. 3) 19 dayspost-PR8 challenge. (B and F) Lung resected from a normal balb/c mouseas a control, (C and G) Lung resected from an AdE/in/−2 mouse (FIG. 3)19 days post-PR8 challenge; each section is a representative of threemice, (D and H) Lung resected from an AdNC/in/−2 mouse (FIG. 3) 19 dayspost-PR8 challenge; each section is a representative of three mice. Lungsections were examined on a Zeiss Axioskop2 plus microscope using a 2×(A-D) or a 10× (E-H) objective lens in conjunction with an Axiocamdigital camera.

FIG. 7 depicts PR8 titers in lungs post-challenge. AdE particles(1.2×10⁸ ifu per 50 ml) were i.n, instilled into mice on day −2 followedby challenging control and AdE-exposed mice with 4.6×10⁶ pfu of PR8 onday 0. Day 5, PR8 titers in lungs resected from control mice 5 dayspost-PR8 challenge; AdE-Day 5, PR8 titers in lungs resected fromAdE-exposed mice 5 days post-PR8 challenge; Day 7, PR8 titers in lungsresected from control mice 7 days post-PR8 challenge; AdE-Day 7, PR8titers in lungs resected from AdE-exposed mice 7 days post-PR8challenge; triangle and circle, log₂(pfu of PR8)/g lung in individualmice; bar, geometric mean of PR8 titers in lungs. No PR8 titers weredetected in lungs resected from control mice that were not challengedwith PR8. The difference between Day 7 and AdE-Day 7 reached statisticalsignificance (by one-way ANOVA with Turkey's multiple comparisonpost-tests).

FIG. 8 depicts protection against lethal challenge by the pandemic CA04in mice. AdE or, AdNC.H1.1 particles (2.5×10⁸ ifu per 50 μl) were i.n.instilled into mice at varying time points followed by CA04 challenge.AdE/in/−22, i.n. administration of AdE on day −22; AdNC/in/−22, i.n.administration of AdNC.H1.1 on day −22; placebo control, i.n.administration of 50 μl saline on day −22; animals were challenged byi.n. instillation of 3×LD₅₀ of the pandemic CA04 on day 0; other,symbols and protocols are the same as those described in FIG. 3 legend.

FIG. 9 depicts architecture of adenovirus. (A) Structure of an Adparticle. Ad is an icosahedral, non-enveloped DNA virus. Its tightlycoiled DNA genome is packaged inside a hexagonal protein capsid. (B)Layout of the ΔE1E3 Ad5 vector. The ΔE1E3 Ad5 vector has been widelyused in a large number of gene therapy as well as vaccine trials. Itshigh immunogenicity was considered a hurdle for re-administration;however, the problem has been lessened by recent evidence showing thatΔE1E3 Ad5-vectored nasal vaccines can bypass pre-existing Ad5 immunity.Ad: Adenovirus; LITR: Left inverted terminal repeat; Promoter: A commonpromoter to drive transgene expression is the cytomegalovirus earlypromoter; PolyA+: A common polyadenylation site is the SV40polyadenylation signal; RITR: Right inverted terminal repeat.

FIG. 10 depicts a pedigree chart of bioengineered non-replicatingadenovirus vectors. Both human and animal Ad have been bioengineeredinto a wide variety of non-replicating Ad vectors for the delivery ofexogenous genes into animal and human subjects. Ad: Adenovirus.

FIG. 11 depicts a manufacturing process for generating Adenoviralvectored vaccine.

FIG. 12 depicts seamless protection conferred by intranasaladministration of an Ad5-vectored drug-vaccine duo. It was recentlydemonstrated that intranasal instillation of AdE (an empty ΔE1E3 Ad5particle without transgene) or AdNC.H1.1 (a ΔE1E3 Ad5 vector encodingthe A/New Caledonia/20/99 HA1 domain) can confer nearly immediateprotection of mice against live influenza virus challenge [Zhang et al.PLoS ONE 6, e22605 (2011)]. The AdE-induced prophylactic therapypersisted in mice for at least 22 days, with partial decline of potencyobserved 47 days post-AdE administration. The AdNC.H1.1-inducedprotection was solid after 47 days. Solid timeframe of solid protection;dashed line: timeframe of partial protection. Since AdE-induced completeprotection was observed for 22 days whereas partial protection wasobserved 47 days post-administration, it was assumed that the drugeffects of DVD started declining after 22 days, as shown by the dashedline following the solid line. It was reported that an Ad5-vectoredvaccine can elicit protective immunity as early as 2 weekspost-immunization [Boyer et al. Hum. Gene Ther. 16, 157-168 (2005)] asshown by a solid line starting on day 14 for the DVDs vaccine effectswhen Ad5 particles were inoculated on day 0. Results show that seamlessprotection against influenza may be achieved in mice by intranasaladministration of an Ad5-vectored DVD since protective immunity can beelicited by the vaccine before the drug effects decline. Ad: Adenovirus;DVD: Drug-vaccine duo.

FIG. 13 depicts Ad5-vectored nasal vaccine protected mice against theA/VN/1203/04 (H5N1) avian influenza virus. Mice were immunized i.n. onDay 0 and challenged with A/VN/1203/04 (H5N1) at a dose of 10×MLD₅₀(10^(4.4) EID₅₀) at SRI on Day 63. HA, Ad encoding HA1+HA2; HA1 Adencoding HA1; E7, 10⁷ vp; E10, 10¹⁰ vp; HI, GMT of serum H1 titers onDay 49.

FIG. 14 depicts an Ad5-vectored nasal vaccine protected ferrets againstthe A/VN/1203/04 (H5N1) avian influenza virus. Ferrets were immunizedi.n. on Day 0; and challenged with A/VN/1203/04 at a dose of 10 FLD₅₀(10² EID₅₀) at SRI on Day 56. HA, Ad encoding HA1+HA2; HA1, Ad encodingHA1; E10, 10¹⁰ vp; HI, GMT of serum HI titers on Day 51.

FIG. 15 depicts prophylactic anthrax therapy by intranasal instillationof adenovirus particles shortly before spore challenge with 1×10⁵ cfu(˜25×LD₅₀) of Bacillus anthracia Sterne spores. AdVAV/−2, AdVAVparticles i.n. instilled 2 days prior to challenge at a dose of 1.3×10⁸ifu; AdE/−2, AdE particles i.n. instilled 2 days prior to challenge at adose of 1.3×10⁸ ifu; AdE*/−2, AdE particles i.n. instilled 2 days priorto challenge at a dose of 1.3×10⁶ ifu (100-fold dilution in PBS);AdE/−1, AdE particles i.n. instilled 1 day prior to challenge at a doseof 1.3×10⁸ ifu; Control, untreated control mice; numbers in parenthesesrepresent the number of animals in each group.

FIG. 16 depicts post-exposure anthrax therapy by i.n. instillation ofAdVAV particles. AdVAV/D−2, AdVAV particles i.n. instilled 2 days priorto challenge at a dose of 1.3×10⁸ ifu; AdVAV/D0, AdVAV particles i.n.instilled 1 hour post-challenge at a dose of 1.3×10⁸ ifu;AdVAV/Cipro/D0, AdVAV particles i.n. instilled 1 hour post-challenge ata dose of 1.3×10⁸ ifu in conjunction with i.p. injection ofciprofloxacin; Cipro/D0, i.p. injection of ciprofloxacin; Control,untreated control mice without treatments prior to challenge; numbers inparentheses represent the number of animals in each group.

FIG. 17 depicts the effect of AdE administered intranasally on RSV-Tracynasal wash as well as lung lavage virus titers on Day +4. Group 1: 6 CRprophylactically (day −2) treated intranasally with vehicle (A195buffer), Group 2: 6 CR prophylactically (day −30) treated intranasallywith 2.4×10⁸ ifu of AdE, Group 3: 6 CR prophylactically (day −2) treatedintranasally with 2.4×10⁸ ifu of AdE, Group 4: 6 CR prophylactically(days −30 and −2) treated intranasally with 2.4×10⁸ ifu of AdE duringeach treatment cycle (prime/boost) and Group 5: 6 CR prophylactically(−5 h) treated intranasally with 2.4×10⁸ ifu of AdE.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based, in part, on the inventor's discovery that asquickly as one day post administration of an empty Ad vector (E1/E3deleted with no insert) mice are protected from a flu challenge. Themechanism for this protection is currently unknown, but it is a verybroad based protection. Mice were protected from a seasonal fluchallenge, swine flu challenge, bird flu challenge, RSV challenge, andeven an anthrax challenge. This protection lasts from about one day toabout 47 days. Wild-type Ad controls did not provide any protection andvaccines given intramuscularly did not provide any protection. Theprotection occurred even when a gene was inserted into the E1 region,although it appears that there was some interference when the gene was aflu HA gene but interestingly, there was an enhanced protection when thegene was an anthrax protective antigen gene. In addition to mice, cottonrats were protected against RSV challenges following intranasaladministration of AdE particles either 2 days or 30 days prior tochallenge.

Embodiments of the invention that employ adenovirus recombinants, mayinclude E1-defective, E3-defective, and/or E4-defective adenovirusvectors. The E1 mutation raises the safety margin of the vector becauseE1-defective adenovirus mutants are replication incompetent innon-permissive cells. The E3 mutation enhances the immunogenicity of theantigen by disrupting the mechanism whereby adenovirus down-regulatesMHC class I molecules. The E4 mutation reduces the immunogenicity of theadenovirus vector by suppressing the late gene expression. Specificsequence motifs such as the RGD motif may be inserted into the H-I loopof an adenovirus vector to enhance its infectivity. An adenovirusrecombinant is constructed by cloning specific transgenes or fragmentsof transgenes into any of the adenovirus vectors such as those describedabove.

Generation of transgene-free ΔE1E3 Ads empty particles may be carriedout as described Tang D C, Zhang J, Toro it, Shi Z, Van Kampen K R(2009) Adenovirus as a carrier for the development of influenzavirus-free avian influenza vaccines, Expert Rev Vaccines 8: 469-481.

The term “viral vector” as used herein includes but is not limited toretroviruses, adenoviruses, adeno-associated viruses, alphavirus, andherpes simplex virus.

The adenovirus may be any adenovirus, such as but not limited to, abovine adenovirus, a canine adenovirus, a non-human primate adenovirus,a chicken adenovirus, or a porcine or swine adenovirus.

The term “human adenovirus” as used herein is intended to encompass allhuman adenoviruses of the Adenoviridae family, which include members ofthe Mastadenovirus genera. To date, over fifty-one human serotypes ofadenoviruses have been identified (see, e.g., Fields et al., Virology 2,Ch. 67 (3d ed., Lippincott-Raven Publishers)). The adenovirus can be ofserogroup A, B, C, E, or F. The human adenovirus can be a serotype 1 (Ad1), serotype 2 (Ad2), serotype 3 (Ad3), serotype 4 (Ad4), serotype 6(Ad6), serotype 7 (Ad7), serotype 8 (Ad8), serotype 9 (Ad9), serotype 10(Ad10), serotype 11 (Ad11), serotype 12 (Ad12), serotype 13 (Ad13),serotype 14 (Ad14), serotype 15 (Ad15), serotype 16 (Ad16), serotype 17(Ad17), serotype 18 (Ad18), serotype 19 (Ad19), serotype 19a (Ad19a),serotype 19p (Ad19p), serotype 20 (Ad20), serotype 21 (Ad21), serotype22 (Ad22), serotype 23 (Ad23), serotype 24 (Ad24), serotype 25 (Ad25),serotype 26 (Ad26), serotype 27 (Ad27), serotype 28 (Ad28), serotype 29(Ad29), serotype 30 (Ad30), serotype 31 (Ad31), serotype 32 (Ad32),serotype 33 (Ad33), serotype 34 (Ad34), serotype 35 (Ad35), serotype 36(Ad36), serotype 37 (Ad37), serotype 38 (Ad38), serotype 39 (Ad39),serotype 40 (Ad40), serotype 41 (Ad41), serotype 42 (Ad42), serotype 43(Ad43), serotype 44 (Ad44), serotype 45 (Ad45), serotype 46 (Ad46),serotype 47 (Ad47), serotype 48 (Ad48), serotype 49 (Ad49), serotype 50(Ad50), serotype 51 (Ad 51), or preferably, serotype 5 (Ad5), but arenot limited to these examples.

Also contemplated by the present invention are receptor-binding ligands,recombinant vectors, drug-vaccine compositions, and recombinantadenoviruses that can comprise subviral particles from more than oneadenovirus serotype. For example, it is known that adenovirus vectorscan display an altered tropism for specific tissues or cell types(Havenga, M. J. E. et al., 2002), and therefore, mixing and matching ofdifferent adenoviral capsids, i.e., fiber, or penton proteins fromvarious adenoviral serotypes may be advantageous. Modification of theadenoviral capsids, including fiber and penton can result in anadenoviral vector with a tropism that is different from the unmodifiedadenovirus. Adenovirus vectors that are modified and optimized in theirability to infect target cells can allow for a significant reduction inthe therapeutic or prophylactic dose, resulting in reduced local anddisseminated toxicity.

Viral vector gene delivery systems are commonly used in gene transferand gene therapy applications. Different viral vector systems have theirown unique advantages and disadvantages. Viral vectors that may be usedto express the pathogen-derived ligand of the present invention includebut are not limited to adenoviral vectors, adeno-associated viralvectors, alphavirus vector, herpes simplex viral vector, and retroviralvectors, described in more detail below.

Adenovirus vectors have many characteristics which are ideal for genedelivery, especially delivery into the respiratory tract. Examples ofthese characteristics include:

-   -   (a) ability of adenovirus vectors to transduce both mitotic and        postmitotic cells in situ;    -   (b) existing technology to prepare stocks containing high titers        of virus [greater than 10¹² ifu (infectious units) per ml] to        transduce cells in situ at high multiplicity of infection (MOI);    -   (c) inhalation of adenovirus is in compliance with evolutionary        medicine (Tang and Van Kampen, 2008);    -   (d) potency of an intranasally-administered adenovirus vector        may not be interfered by preexisting immunity to adenovirus        (Hoelscher et al., 2006; Shi et al., 2001; Van Kampen et al.,        2005); while not wishing to be bound by theory, this may be        attributed to the high efficiency of gene delivery, high level        of transgene expression, and high degree of antigen presentation        along the mucosal barrier in the respiratory tract;    -   (e) capability of adenovirus to induce high levels of transgene        expression (at least as an initial burst); and    -   (f) ease with which replication-defective adenovirus vectors can        be bioengineered.

Additional general features of adenoviruses are that the biology of theadenovirus is characterized in detail; the adenovirus is not associatedwith severe human pathology; the adenovirus is extremely efficient inintroducing its DNA into the host cell; the adenovirus can infect a widevariety of cells and has a broad host range; the adenovirus can beproduced in large quantities with relative ease; and the adenovirus canbe rendered replication defective and/or non-replicating by deletions inthe early region 1 (“E1”) of the viral genome.

Reference is made to U.S. Pat. No. 5,990,091 issued Nov. 23, 1999, Einatet al. or Quark Biotech, Inc., WO 99/60164, published Nov. 25, 1999 fromPCT/US99/11066, filed May 14, 1999, Fischer or Rhone Merieux, Inc.,WO98/00166, published Jan. 8, 1998 from PCT/US97/11486, filed Jun. 30,1997 (claiming priority from U.S. application Ser. No. 08/675,556 andSer. No. 08/675,566), van Ginkel et al., J. Immunol 159(2):685-93 (1997)(“Adenoviral gene delivery elicits distinct pulmonary-associated Thelper cell responses to the vector and to its transgene”), andOsterhaus et al., Immunobiology 184(2-3):180-9′2 (1992) (“Vaccinationagainst acute respiratory virus infections and measles in man”), forinformation concerning expressed gene products, antibodies and usesthereof, vectors for in vivo and in vitro expression of exogenousnucleic acid molecules, promoters for driving expression or foroperatively linking to nucleic acid molecules to be expressed, methodand documents for producing such vectors, compositions comprising suchvectors or nucleic acid molecules or antibodies, dosages, and modesand/or routes of administration (including compositions for nasaladministration), inter alia, which can be employed in the practice ofthis invention; and thus, U.S. Pat. No. 5,990,091 issued Nov. 23, 1999,Einat et al. or Quark Biotech, Inc., WO 99/60164, published Nov. 25,1999 from PCT/US99/11066, filed May 14, 1999, Fischer or Rhone Merieux,Inc., WO98/00166, published Jan. 8, 1998 from PCT/US97/11486, filed Jun.30, 1997 (claiming priority from U.S. application Ser. No. 08/675,556and Ser. No. 08/675,566), van Ginkel et al., J. Immunol 159(2):685-93(1997) (“Adenoviral gene delivery elicits distinct pulmonary-associatedT helper cell responses to the vector and to its transgene”), andOsterhaus et al., Immunobiology 184(2-3):180-92 (1992) (“Vaccinationagainst acute respiratory virus infections and measles in man”) and alldocuments cited or referenced therein and all documents cited orreferenced in documents cited in each of U.S. Pat. No. 5,990,091 issuedNov. 23, 1999, Einat et al. or Quark Biotech, Inc., WO 99/60164,published Nov, 25, 1999 from PCT/US99/11066, filed May 14, 1999, Fischeror Rhone Merieux, Inc., WO98/00166, published Jan. 8, 1998 fromPCT/US97/11486, filed Jun. 30, 1997 (claiming priority from U.S.application Ser. No. 08/675,556 and Ser. No. 08/675,566), van Ginkel etal., J. Immunol 159(2):685-93 (1997) (“Adenoviral gene delivery elicitsdistinct pulmonary-associated. T helper cell responses to the vector andto its transgene”), and Osterhaus et al., Immunobiology 184(2-3):180-92(1992) (“Vaccination against acute respiratory virus infections andmeasles in man”) are hereby incorporated herein by reference.Information in U.S. Pat. No. 5,990,091 issued Nov. 23, 1999, WO99/60164, WO98/00166, van Ginkel et al., J. Immunol 159(2):685-93(1997), and Osterhaus et al., Immunobiology 184(2-3): 180-92 (1992) canbe relied upon for the practice of this invention (e.g., expressedproducts, antibodies and uses thereof, vectors for in vivo and in vitroexpression of exogenous nucleic acid molecules, exogenous nucleic acidmolecules encoding epitopes of interest or antigens or therapeutics andthe like, promoters, compositions comprising such vectors or nucleicacid molecules or expressed products or antibodies, dosages, interalia).

It is noted that immunological products and/or antibodies and/orexpressed products obtained in accordance with this invention can beexpressed in vitro and used in a manner in which such immunologicaland/or expressed products and/or antibodies are typically used, and thatcells that express such immunological and/or expressed products and/orantibodies can be employed in in vitro and/or ex vivo applications,e.g., such uses and applications can include diagnostics, assays, exvivo therapy (e.g., wherein cells that express the gene product and/orimmunological response are expanded in vitro and reintroduced into thehost or animal), etc., see U.S. Pat. No. 5,990,091, WO 99/60164 and WO98/00166 and documents cited therein.

Further, expressed antibodies or gene products that are isolated fromherein methods, or that are isolated from cells expanded in vitrofollowing herein administration methods, can be administered incompositions, akin to the administration of subunit epitopes or antigensor therapeutics or antibodies to induce immunity, stimulate atherapeutic response and/or stimulate passive immunity. The quantity tobe administered will vary for the patient (host) and condition beingtreated and will vary from one or a few to a few hundred or thousandmicrograms, e.g., 1 μg to 1 mg, from about 100 ng/kg of body weight to100 mg/kg of body weight per day and preferably will be from 10 pg/kg to10 mg/kg per day.

A vector can be administered to a patient or host in an amount toachieve the amounts stated for gene product (e.g., epitope, antigen,therapeutic, and/or antibody) compositions. Of course, the inventionenvisages dosages below and above those exemplified herein, and for anycomposition to be administered to an animal or human, including thecomponents thereof, and for any particular method of administration, itis preferred to determine therefor: toxicity, such as by determining thelethal dose (LD) and LD₅₀ in a suitable animal model e.g., rodent suchas mouse; and, the dosage of the composition(s), concentration ofcomponents therein and timing of administering the composition(s), whichelicit a suitable response, such as by titrations of sera and analysisthereof, e.g., by ELISA and/or seroneutralization analysis. Suchdeterminations do not require undue experimentation from the knowledgeof the skilled artisan, this disclosure and the documents cited herein.And, the invention also comprehends sequential administration ofinventive compositions or sequential performance of herein methods,e.g., periodic administration of inventive compositions such as in thecourse of therapy or treatment for a condition and/or boosteradministration of immunological compositions and/or in prime boostregimens; and, the time and manner for sequential administrations can beascertained without undue experimentation.

The dosage of the adenovirus of the present invention may be from about10⁶ ifu to about 10¹⁶ ifu. The dosage may be about 10⁶ ifu, about 10⁷ifu, about 10⁸ ifu, about 10⁹ ifu or about 10¹⁰ ifu. In an advantageousembodiment, the dosage is about 10⁶ ifu, about 10⁷ ifu or about 10⁸ ifu.

In a particularly advantageous embodiment, multiple dosages of theadenovirus of the present invention. In a particularly advantageousembodiment, about two doses are administered. In an advantageousembodiment, the doses are administered about 20 days apart, about 25days apart, about 30 days apart, about 35 days apart, about 40 daysapart, about 45 days apart, about 50 days apart, about 55 days apart,about 60 days apart or about 65 days apart. Advantageously, the dosesare administered about 40 days apart, about 41 days apart, about 42 daysapart, about 43 days apart, about 44 days apart, about 45 days apart,about 46 days apart, about 47 days apart, about 48 days apart, about 49days apart or about 50 days apart,

Further, the invention comprehends compositions and methods for makingand using vectors, including methods for producing gene products and/orimmunological products and/or antibodies in vivo and/or in vitro and/orex vivo (e.g., the latter two being, for instance, after isolationtherefrom from cells from a host that has had a non-invasiveadministration according to the invention, e.g., after optionalexpansion of such cells), and uses for such gene and/or immunologicalproducts and/or antibodies, including in diagnostics, assays, therapies,treatments, and the like. Vector compositions are formulated by admixingthe vector with a suitable carrier or diluent; and, gene product and/orimmunological product and/or antibody compositions are likewiseformulated by admixing the gene and/or immunological product and/orantibody with a suitable carrier or diluent; see, e.g., U.S. Pat. No.5,990,091, WO 99/60164, WO 98/00166, documents cited therein, and otherdocuments cited herein, and other teachings herein (for instance, withrespect to carriers, diluents and the like).

In an advantageous embodiment, the vector expresses a gene encoding aninfluenza antigen, a RSV antigen, a HIV antigen, a SIV antigen, a HPVantigen, a HCV antigen, a HBV antigen, a CMV antigen or a Staphylococcusantigen. The influenza may be swine influenza, seasonal influenza, avianinfluenza, H1N1 influenza or H5N1 influenza.

In another advantageous embodiment, the vector expresses a gene whichencodes influenza hemagglutinin, influenza nuclear protein, influenzaM2, tetanus toxin C-fragment, anthrax protective antigen, anthrax lethalfactor, rabies glycoprotein, HBV surface antigen, HIV gp 120, HW gp 160,human carcinoembryonic antigen, malaria CSP, malaria SSP, malaria MSP,malaria pfg, mycobacterium tuberculosis HSP or a mutant thereof.

In an embodiment of the invention, the immune response in the animal isinduced by genetic vectors expressing genes encoding antigens ofinterest in the animal's cells. The antigens of interest may be selectedfrom any of the antigens described herein.

In another embodiment of the method, the animal's cells are epidermalcells. In another embodiment of the method, the immune response isagainst a pathogen or a neoplasm. In another embodiment of the method,the genetic vector is used as a prophylactic vaccine or a therapeuticvaccine. In another embodiment of the invention, the genetic vectorcomprises genetic vectors capable of expressing an antigen of interestin the animal's cells. In a further embodiment of the method, the animalis a vertebrate.

With respect to exogenous DNA for expression in a vector (e.g., encodingan epitope of interest and/or an antigen and/or a therapeutic) anddocuments providing such exogenous DNA, as well as with respect to theexpression of transcription and/or translation factors for enhancingexpression of nucleic acid molecules, and as to terms such as “epitopeof interest”, “therapeutic”, “immune response”, “immunologicalresponse”, “protective immune response”, “immunological composition”,“immunogenic composition”, and “vaccine composition”, inter alia,reference is made to U.S. Pat. No. 5,990,091 issued Nov. 23, 1999, andWO 98/00166 and WO 99/60164, and the documents cited therein and thedocuments of record in the prosecution of that patent and those PCTapplications; all of which are incorporated herein by reference. Thus,U.S. Pat. No. 5,990,091 and WO 98/00166 and WO 99/60164 and documentscited therein and documents or record in the prosecution of that patentand those PCT applications, and other documents cited herein orotherwise incorporated herein by reference, can be consulted in thepractice of this invention; and, all exogenous nucleic acid molecules,promoters, and vectors cited therein can be used in the practice of thisinvention. In this regard, mention is also made of U.S. Pat. Nos.6,004,777, 5,997,878, 5,989,561, 5,976,552, 5,972,597, 5,858,368,5,863,542, 5,833,975, 5,863,54 5,843,456, 5,766,598, 5,766,597,5,762,939, 5,756,102, 5,756,101, 5,494,807.

In another embodiment of the invention, the animal is advantageously avertebrate such as a mammal, bird, reptile, amphibian or fish; moreadvantageously a human, or a companion or domesticated or food-producingor feed-producing or livestock or game or racing or sport animal such asa cow, a dog, a cat, a goat, a sheep or a pig or a horse, or even fowlsuch as turkey, ducks or chicken. In an especially advantageous anotherembodiment of the invention, the vertebrate is a human.

In another embodiment of the invention, the genetic vector is a viralvector, a bacterial vector, a protozoan vector, a retrotransposon, atransposon, a virus shell, or a DNA vector. In another embodiment of theinvention, the viral vector, the bacterial vector, the protozoan vectorand the DNA vector are recombinant vectors. In another embodiment of theinvention, the immune response is against influenza A. In anotherembodiment of the invention, the immune response against influenza A isinduced by the genetic vector expressing a gene encoding an influenzahemagglutinin, an influenza nuclear protein, an influenza M2 or afragment thereof in the animal's cells. In another embodiment of theinvention, the genetic vector is selected from the group consisting ofviral vector and plasmid DNA.

In another embodiment of the invention, the genetic vector is anadenovirus. In another embodiment of the invention, the adenovirusvector is defective in its E1 region. In another embodiment of theinvention, the adenovirus vector is defective in its E3 region. Inanother embodiment of the invention, the adenovirus vector is defectivein its E1 and/or E3 regions. In another embodiment of the invention, theDNA is in plasmid form. In another embodiment of the invention, thecontacting step further comprises disposing the genetic vectorcontaining the gene of interest on a delivery device and applying thedevice having the genetic vector containing the gene of interest thereinto the skin of the animal. In another embodiment of the invention, thegenetic vector encodes an irnmunomodulatory gene, as co-stimulatory geneor a cytokine gene. In another embodiment of the invention, the vectorhas all viral genes deleted. In another embodiment of the invention, thegenetic vector induces an anti-tumor effect in the animal. In a furtherembodiment of the invention, the genetic vector expresses an oncogene, atumor-suppressor gene, or a tumor-associated gene.

Representative examples of antigens which can be used to produce animmune response using the methods of the present invention includeinfluenza hemagglutinin, influenza nuclear protein, influenza M2,tetanus toxin C-fragment, anthrax protective antigen, anthrax lethalfactor, rabies glycoprotein, HBV surface antigen, HIV gp 120, HIV gp160, human carcinoembryonic antigen, malaria CSP, malaria SSP, malariaMSP, malaria pfg, and mycobacterium tuberculosis HSP, etc, Mostpreferably, the immune response produces a protective effect againstneoplasms or infectious pathogens.

In another embodiment of the present invention, the vector furthercontains a gene selected from the group consisting of co-stimulatorygenes and cytokine genes. In this method the gene is selected from thegroup consisting of a GM-CSF gene, a B7-1 gene, a B7-2 gene, aninterleukin-2 gene, an interleukin-12 gene and interferon genes.

The recombinant vectors and methods of the present invention can be usedin the treatment or prevention of various respiratory pathogens. Suchpathogens include, but are not limited to, influenza virus, severe acuterespiratory syndrome-associated coronavirus (SARS-CoV), human rhinovirus(HRV), and respiratory syncytial virus (RSV).

In addition, the present invention comprehends the use of more thantherapeutic ligand, immunogen or antigen in the vectors and methodsdisclosed herein, delivered either in separate recombinant vectors, ortogether in one recombinant vector so as to provide a multivalentvaccine or immunogenic composition that stimulates or modulatesimmunogenic response to one or more influenza strains and/or hybrids.Further, the present invention encompasses the use of a therapeuticligand, immunogen or antigen from more than one pathogen in the vectorsand methods disclosed herein, delivered either in separate recombinantvectors, or together in one recombinant vector.

Embodiments of the invention that use DNA/adenovirus complexes can havethe plasmid DNA complexed with adenovirus vectors utilizing a suitableagent therefor, such as either PEI (polyethylenimine) or polylysine. Theadenovirus vector within the complex may be either “live” or “killed” byUV irradiation. The UV-inactivated adenovirus vector as areceptor-binding ligand and an endosomolysis agent for facilitatingDNA-mediated transfection (Cotten et al., 1992) may raise the safetymargin of the vaccine carrier. The DNA/adenovirus complex is used totransfect epidermal cells of a vertebrate in a non-invasive mode for useas an immunizing agent.

Genetic vectors provided by the invention can also code forimmunomodulatory molecules which can act as an adjuvant to provoke ahumoral and/or cellular immune response. Such molecules includecytokines, co-stimulatory molecules, or any molecules that may changethe course of an immune response. One can conceive of ways in which thistechnology can be modified to enhance still further the immunogenicityof antigens.

In terms of the terminology used herein, an immunologically effectiveamount is an amount or concentration of the genetic vector encoding thegene of interest, that, when administered to an animal, produces animmune response to the gene product of interest.

Various epitopes, antigens or therapeutics may be delivered topically byexpression thereof at different concentrations, Generally, usefulamounts for adenovirus vectors are at least approximately 100 pfu andfor plasmid DNA at least approximately 1 ng of DNA. Other amounts can beascertained from this disclosure and the knowledge in the art, includingdocuments cited and incorporated herein by reference, without undueexperimentation.

The methods of the invention can be appropriately applied to preventdiseases as prophylactic vaccination or treat diseases as therapeuticvaccination.

The vaccines of the present invention can be administered to an animaleither alone or as part of an immunological composition.

Beyond the human vaccines described, the method of the invention can beused to immunize animal stocks. The term animal means all animalsincluding humans. Examples of animals include humans, cows, dogs, cats,goats, sheep, horses, pigs, turkey, ducks and chicken, etc. Since theimmune systems of all vertebrates operate similarly, the applicationsdescribed can be implemented in all vertebrate systems.

The present invention also encompasses combinations of vectors, inparticular adenovirus vectors. For example, an empty adenovector (E1/E3deleted with no insert) may be sequentially or simultaneouslyadministered to a patient in need thereof along with another vector,such as an adenovector, which may be E1/E3 deleted with an insert, suchas an exogenous gene as herein described. Without being bound by theory,the empty adenovector (E1/E3 deleted with no insert) may initiallyelicit a rapid immune response wherein a vector expressing an exogeneousgene, such as an antigen or epitope, may elicit an additional protectiveresponse.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims. The presentinvention will be further illustrated in the following Examples whichare given for illustration purposes only and are not intended to limitthe invention in any way.

EXAMPLES Example 1 Adenovirus Particle as a Broad-Spectrum Drug AgainstRespiratory Pathogens

Although vaccination is an effective approach for preventing infectiousdiseases when administered weeks or months in advance, it is too slow toprotect animals or people who are at immediate risk. An agent capable ofreducing the severity of an infection when taken shortly before or afteran infection is of paramount importance in public health. Tamiflu(oseltamivir phosphate) and Relenza (zanamivir) have proven effective inpreventing influenza virus infections; however, these neuraminidaseinhibitors may generate drug-resistant influenza virus strains over time(Poland et al., 2009). Similar to viruses, drug-resistant bacteria arealso commonly generated by overuse of drugs (Davies and Davies, 2010).It is thus urgent to develop additional drugs because medical personnelwill have the option to use another drug in the pipeline for arrestingpathogens when one drug in use is impaired by drug resistance.

Applicant has demonstrated that intranasal instillation ofE1/E3-defective adenovirus (Ad) particles 1-2 days prior to intranasalchallenge with a lethal dose of influenza virus could confer rapidprotection against influenza in mice (FIG. 1). Applicant has also shownthat intranasal administration of Ad 1-2 days prior to intranasalchallenge with a lethal dose of Bacillus anthracia Sterne spores wasalso able to protect mice against anthrax (FIG. 2 and 15). It isconceivable that intranasal administration of Ad rapidly induces anarray of reactions that impede growth of other microbes in therespiratory tract.

Since the Ad has been bioengineered into a non-replicating vaccinecarrier with an excellent safety profile in animals and humans (Tang etal., 2009), it is conceivable that an Ad vector encoding apathogen-derived antigen may be developed into a drug vaccine duo (DVD),which is able to confer rapid and broad protection against a variety ofpathogens before adaptive immunity is induced; followed by elicitationof pathogen specific protective immunity as a vaccine; in a singlepackage. FIGS. 1 and 2 show that not only the transgene-free Ad backbone(AdE) could confer rapid protection against viruses and bacteria as adrug, but also its counterparts encoding pathogen derived antigens wereable to arrest pathogens as a drug.

Methods of FIG. 1. Purified AdNC.H1.1 vectors encoding the HA1 ofA/NC/20/99 (H1N1) influenza virus and its transgene-free counterpart(AdE) were administered dropwise in a volume of 0.05 ml into the nostrilof each young BALM mouse (2 months old) using a mechanical pipet asdescribed (Shi et al., 2001) prior to challenge. One or two days post-Adadministration, mice were challenged intranasally with a lethal dose(0.2 HA units) of the A/PR/8/34 (H1N1) influenza virus and monitoreddaily for survival.

Results of FIG. 1. Seventy percent of mice (7/10) were protected againsta lethal dose of live A/PR/8/34 influenza virus by intranasalinstillation of the AdNC.H1.1 vector at a dose of 1.7×10⁸ infectiousunits (ifu) 2 days prior to challenge (AdEin−2); 100% of mice (10/10)were protected against influenza by intranasal instillation of the AdEvector at a dose of 1.7×10⁶ ifu 2 days prior to challenge (AdEin−2); 20%of mice (2/10) were protected against influenza by intranasalinstillation of the AdE vector at a dose of 1.7×10⁶ ifu 2 days prior tochallenge (AdE*in−2); 90% of mice (9/10) were protected againstinfluenza by intranasal instillation of the AdE vector at a dose of1.7×10⁸ ifu 1 day prior to challenge (AdEin−1); all control mice (Naivecontrol) died within 10 days post-challenge. The data was plotted as %survival versus days after challenge. Numbers in parentheses representthe number of animals in each group.

Significance of FIG. 1. Protection of animals against influenza byintranasal administration of an Ad vector 1 or 2 days prior to liveinfluenza virus challenge shows that the Ad particle is able to rapidlyinduce an anti-viral state in the respiratory tract. Since Ad vectorsencoding influenza virus. HA have been developed into influenza vaccines(Hoelscher et al., 2006; Tang et al, 2009; Van Kampen et al., 2005) andthe AdNC.H1.1 vector encoding the A/NC/20/99 HA still conferred rapidprotection as an influenza drug before adaptive immunity was elicited,there is compelling evidence that this regimen represents a drug-vaccineduo (DVD) that can confer broad and rapid protection as an anti-viraldrug followed by elicitation of protective immunity as an anti-viralvaccine, in a single package.

Methods of FIG. 2. Purified AdPA83 vectors encoding the Bacillusanthracis protective antigen and its transgene-free counterpart (AdE)were administered dropwise in a volume of 0.05 ml into the nostril ofeach young A/J mouse (2 months old) using a mechanical pipet asdescribed (Shi et al., 2001) prior to challenge. One or two days post-Adadministration, mice were challenged intranasally with a lethal dose[1×10⁵ colony-forming units (cfu)] of B. anthracis Sterne spores andmonitored daily for survival.

Results of FIG. 2. Sixty-seven percent of mice (6/9) were protectedagainst a lethal dose of anthrax spores by intranasal instillation ofthe AdPA83 vector at a dose of 1.3×10⁸ ifu 2 days prior to challenge(AdPAin−2); 30% of mice (3/10) were protected against anthrax byintranasal instillation of the AdE vector at a dose of 1.3×10⁸ ifu 2days prior to challenge (AdEin−2); no mice (0/9) were protected againstanthrax by intranasal instillation of the AdE vector at a dose of1.3×10⁶ ifu 2 days prior to challenge (AdE*in−2); 22% of mice (2/9) wereprotected against anthrax by intranasal instillation of the AdE vectorat a dose of 1.3×10⁸ ifu 1 day prior to challenge (AdEin−1); all controlmice (Naive control) died within 4 days post-challenge. The data wasplotted as % survival versus days after challenge. Numbers inparentheses represent the number of animals in each group.

Significance of FIG. 2. Protection of animals against anthrax byintranasal administration of an Ad vector 1 or 2 days prior to anthraxspore challenge shows that the Ad particle is able to rapidly induce ananti-bacterial state in the respiratory tract. Since Ad vectors encodingPA have been developed into anthrax vaccines (McConnell et al., 2007)and the AdPA83 vector encoding the PA still conferred rapid protectionas an anthrax drug before adaptive immunity was elicited, there iscompelling evidence that this regimen represents a drug-vaccine duo(DVD) that can confer rapid protection as an anti-bacterial drugfollowed by elicitation of protective immunity as an anti-bacterialvaccine, in a single package.

REFERENCES

-   Davies, J., and Davies, D. (2010). Origins and evolution of    antibiotic resistance. Microbiol Mol Bioi Rev 74,417-433.-   Hoelscher, M. A., Garg, S., Bangari, D. S., Belser, J. A., Lu, X.,    Stephenson, I., Bright, R. A., Katz, J. M., Mittal, S. K., and    Sambhara, S. (2006). Development of adenoviral-vector-based pandemic    influenza vaccine against antigenically distinct human H5N1 strains    in mice. Lancet 367,475-481.-   McConnell, M. J., Hanna, P. C., and Imperiale, M. J. (2007).    Adenovirus-based prime-boost immunization for rapid vaccination    against anthrax. Mol Ther 15,203-210.-   Poland, G. A., Jacobson, R. M., and Ovsyannikova, I. G. (2009).    Influenza virus resistance to antiviral agents: a plea for rational    use. Clin Infect Dis 48, 1254.1256.-   Shi, Z., Zeng, M., Yang, G., Siegel, F., Cain, L. J., Van Kampen, K.    R., Elmets, C. A., and Tang, D. C. (2001). Protection against    tetanus by needle-free inoculation of adenovirus-vectored nasal and    epicutaneous vaccines. J Virol 75, 11474-11482.-   Tang, D. C., Zhang, J., Toro, H., Shi, Z., and Van Kampen, K. A.    (2009). Adenovirus as a carrier for the development of influenza    virus-free avian influenza vaccines. Expert Rev Vaccines 8, 469-481,-   Van Kampen, K. R., Shi, Z., Gao, P., Zhang, J., Foster, K. W.,    Chen, D. T., Marks, D., Elmets, C. A., and Tang, D. C. (2005),    Safety and immunogenicity of adenovirus-vectored nasal and    epicutaneous influenza vaccines in humans. Vaccine 23, 1029.1036.

Example 2 Adenovirus-Vectored Drug-Vaccine Duo as a Rapid-Response Toolfor Conferring Seamless Protection Against Influenza

Few other diseases exert such a huge toll of suffering as influenza.Applicant reports here that intranasal (i.n.) administration ofE1/E3-defective (ΔE1E3) adenovirus serotype 5 (Ad5) particles rapidlyinduced an anti-influenza state as a means of prophylactic therapy whichpersisted for several weeks in mice. By encoding an influenza virus(IFV) hemagglutinin (HA) HA1 domain, an Ads-HA1 vector conferred rapidprotection as a prophylactic drug followed by elicitation of sustainedprotective immunity as a vaccine for inducing seamless protectionagainst influenza as a drug-vaccine duo (DVD) in a single package. SinceAds particles induce a complex web of host responses, which could arrestinfluenza by activating a specific arm of innate immunity to impede IFVgrowth in the airway, it is conceivable that this multi-prongedinfluenza DVD may escape the fate of drug resistance that impairs thecurrent influenza drugs.

Influenza is a resurging and emerging disease with virtually nopossibility of eradicating the causal virus which triggers seasonal aswell as pandemic influenza. As a zoonotic disease with the potential tosicken both animals and humans [1], a designer IFV can be rapidly^(,)generated by reverse genetics [2] and disseminated by terrorists toravage agriculture, public health, and economy within a targeted region.Even though this highly contagious and potentially fatal disease hasbeen partially controlled by vaccination, the licensed influenza vaccineis difficult to mass-produce [1] and unable to confer timely as well asbroad protection against heterosubtypic IFV strains [3]. Another line ofdefense against influenza is the use of influenza drugs [e.g.,oseltarnivir (Tamiflu); zanamivir (Relenza)]; however, this option islimited by the emergence of drug-resistant IFV due to selection undermutational pressure [4,5].

To develop a rapid-response anti-influenza agent, we serendipitouslydemonstrated that an Ad5-vectored nasal influenza vaccine could conferrapid protection against influenza in a drug-like manner. Areplication-competent adenovirus (RCA)-free Ad5 vector encoding pathogenantigens thus potentially can confer seamless protection against mucosalpathogens as a DVD in a wide variety of clinical settings. RCA-free Ad5vectors can be rapidly mass-produced in serum-free PER.C6 suspensioncells; painlessly mass-administered by nasal spray [1]; followed byelicitation of innate as well as adaptive immune responses in the faceof pre-existing Ad5 immunity. In the case of an influenza DVD, thechance to generate drug-resistant IFV is minimal since Ad5 particlesconceivably induce an anti-influenza state without directly attackingthe IFV. In contrast to a live attenuated IFV vaccine (LAIV), anAd5-vectored DVD is non-replicating and does not reassort with wild IFV.It is expected that nasal spray of an Ad5-vectored influenza. DVD canconfer broad protection against heterosubtypic IFV strains for severalweeks as a prophylactic drug; followed by elicitation of strain-specificprotective immunity as a vaccine for months or even years before thedrug induced protection declines away. This novel regimen may add arapid-response tool to the public health arsenal against influenza andother diseases if the DVD's protective effects should be reproduced inhuman subjects.

The ΔE1E3 Ad5 particle as an anti-influenza agent. The transgene-freeΔE1E3 Ad5 empty (AdE) particle and its counterpart AdNC.H1.1 encodingthe A/New Caledonia/20/99 H1N1 IFV (NC20) HA1 domain were generated inPER.C6 cells as described [1]. As shown in FIG. 3A, i.n. instillation of1.7×10⁸ infectious units (ifu) of AdE 2 days (day −2) prior to challengeprotected 100% (10/10) of mice against a lethal dose of live A/PuertoRico/8/34 H1N1 IFV (PR8); only 20% (2/10) of the animals were protectedwhen AdE's dose was reduced 100-fold to 1.7×10⁶ ifu; and there was noprotection when 1.7×10⁸ ifu of AdE were administered into mice by i.n.instillation 1 day post-PR8 challenge or by i.m. injection on day −2.Insertion of the NC20 HA1 domain into the AdE genome mildly interferedwith ΔE1E3 Ad53 s capacity to induce an anti-influenza state as only 70%(7/10) of animals were protected when 1.7×10⁸ ifu of AdNC.H1.1 were i.n.administered into mice on day −2. Similar to AdE, neither i.n.instillation of 1.7×10⁶ ifu nor i.m. injection of 1.7×10⁸ ifu ofAdNC.H1.1 conferred any protection against PR8 when administered on day−2 (FIG. 3). The protection afforded by i.n. administration of AdE(P,0.0001) or AdNC.H1.1 (P=0.0077) at a dose of 1.7×10⁸ ifu on day −2reached statistical significance when compared to that of the untreatedcontrol group (by Logrank tests).

Intranasal administration of AdE on day −47 (47 days prior to PR8challenge) protected 70% of animals (7/10) showing that the AdE-inducedanti-influenza state could persist for several weeks (FIG. 4A).Intranasal instillation of AdNC.H1.1 on day −47 protected 100% (10/10)of mice (FIG. 4A) presumably due to NC20 HA1-induced adaptive immunitywhich cross-reacted with PR8 even though no serumhemagglutination-inhibition (HI) antibodies to PR8 were detectable(Table 1). Unlike immunization with AdNC.H1.1 on day −47 which elicitedhigh HI antibody titers to NC20 and undetectable titers to PR8,challenge with PR8 induced high HI antibody titers to PR8 and low titersto NC20 in survivors, and administration of either AdE or AdNC.H1.1 onday −2 induced HI titers to neither NC20 nor PR8 (Table 1). Theprotection afforded by i.n. administration of AdNC.H1.1 on day −47(P,0.0001), AdE on day −47 (P=0.0032), AdE double-dose regimen (day −47followed by a booster application on day −2) (P,0.0001), AdE on day −1(P,0.0001) or −2 (P=0.0005) at a dose of 1.2×10⁸ ifu all reachedstatistical significance when compared to that of the untreated controlgroup.

TABLE 1 Serum H1 antibody titers induced by AdNCH1.1 immunization andPR8 challenge. Log₂ [anti- Log₂ [anti- Day of NC20 HI Seroconversion PR8HI Seroconversion serum GMT] to NC20 GMT] to PR8 Immunization ncollection (±SD) (%) (±SD) (%) ^(a)AdNC/in/−2 + 7 19  7.9 (±0.5) 100 8.9(±0.5) 100 PR8 ^(a)AdE/in/−2 + 10 19  5.3 (±0.7) 100 7.5 (±0.6) 100 PR8^(b)AdNC/in/−47 10 −1 10.2 (±1.7) 100 2.3 (±0) 0 ^(b)AdNC/in/−2 10 −1 2.3 (±0) 0 2.3 (±0) 0 ^(b)AdE/in/−2 10 −1  2.3 (±0) 0 2.3 (±0) 0^(b)Untreated 10 −1  2.3 (±0) 0 2.3 (±0) 0 control HI antibodies weremeasured against the respective IFV with titers expressed as GMT on alog₂ scale; a log₂ titer of 2.3 was arbitrarily assigned to samples withundetectable titers; each serum samples was run in triplicate wells;^(a)animals described in FIG. 1. with sera collected 19 days post-PR8challenge; ^(b)animals described in FIG 2. with sera collected 1 dayprior to PR8 challenge. Seroconversion was defined as ≥4-fold rise in HItiter above the preimmune baseline; n, number of animals; GMT, geometricmean titer; SD, standard deviation.

Although several regimens protected mice against influenza-mediatedmortality, the AdE double-dose regimen tended to confer more solidprotection than its single-dose (day −−47 or −2) counterpart as shown byless body weight loss after PR8 challenge even though the difference didnot reach statistical significance (FIG. 5). To induce an anti-influenzastate, it is essential to delete E1 and/or E3 since the E1+/E3+wild-type Ad5 was unable to arrest influenza after i.n. administrationinto mice under identical conditions (FIG. 4B).

Ad5-induced protection of the lung against influenza. As shown by lunghistopathology after PR8 challenge, i.n. administration of AdE orAdNC.H1.1 on day −2 protected mice against influenza by preventing thedevelopment of severe lung injuries. Intranasal instillation of PR8without Ad5 protection induced massive pulmonary inflammation 19 dayspost-challenge one-way ANOVA with Turkey's multiple comparisonpost-tests) 7 days post-PR8 challenge.

Protection against a pandemic IFV strain. To demonstrate that ΔE1E3 Ad5particles can protect mice against not only PR8 but also a moreclinically relevant IFV strain, 2.5×10⁸ ifu of AdE or AdNC.H1.1 werei.n. administered into mice followed by challenging animals with alethal dose of the (pandemic 2009 H1N1 swine flu isolateA/California/04/2009 (CA04). As shown in FIG. 8, 100% (10/10) of animalswere protected by i.n. instillation of AdE or AdNC.H1.1 on day −2 andAdNC.H1.1 on day −22; 90% (9/10) were protected by i.n. administrationof AdE on day −22. The protection afforded against CA04 in all theseAd5-exposed groups reached statistical significance when compared tothat of the placebo control group (P,0.0001),

The non-replicating ΔE1E3 Ad5 vector has been bioengineered into a nasalinfluenza vaccine carrier with high potency and excellent safety profile[1]. In addition to the elicitation of protective immunity as a vaccine,we show here that this class of vaccine can also confer prophylactictherapy against influenza before adaptive immunity is elicited. It hasbeen documented that administration of ΔE1E3 Ad5 particles into micerapidly induces the production of a wide array of inflammatory cytokinesand chemokines [6] including type I interferon (IFN-a and IFN-b) [7];impairs lung dendritic cells [8]; activates natural killer cells [9];induces production of the antiviral nitric oxide [10]; triggersmultifaceted interactions between Ad5 and blood proteins, platelets,macrophages, endothelial cells, and respective parenchymal cells [6].Inhibition of Ad5-associated inflammation by Ad5 E1A, E1B, and E3proteins [11] suggests that the E1+/E3+ Ad5's incompetence to induce ananti-influenza state (FIG. 4) may be attributed to suppression ofinflammation, although other mechanisms cannot be excluded since ΔE1E3Ad5 particles induce many immune as well as non-immune responses andsome reactions remain undefined in animals [12]. It is conceivable thatmultiple reactions induced by the ΔE1E3 Ad5 particles may integrate forestablishing an anti-influenza state in the airway, thus creating amultidimensional defense barrier that can hardly be bypassed by an IFV.This hypothesis is supported by the finding that the IFN-α,β receptorprovides protection against influenza in a dispensable manner showingthat animals have evolved overlapping mechanisms to respond to influenza[13]. Furthermore, Balb/c mice challenged in these studies carry adefective allele of the IFN-α,β-induced influenza-resistance factor M×1.[14] implying that the ΔE1E3 Ad5-induced production of type I IFN [7]may not play a major role during the establishment of an anti-influenzastate in this mouse strain.

The finding that i.n. administration of AdE 1 day post-PR8 challenge wasunable to arrest influenza (FIG. 3A) suggests that the !WV may induce apro-influenza state that is not disrupted by the ΔE1E3 Ad5 particle whenthe former enters the airway prior to the latter, similar to theAd5-induced anti-influenza state that cannot be reversed by an TV whenAdE particles were i.n. administered prior to PR8 or CA04 (FIGS. 3-9).To further develop the ΔE1E3 Ad5-based prophylactic drug into apost-exposure influenza drug, it is crucial to characterize theantagonistic reactions induced by the two types of viruses in theairway.

Pre-exposure to Ad5 has been associated with loss of Ad5's potency whenthis vector is i.m. injected [15]. However, emerging evidence shows thatan Ad5-vectored nasal vaccine can bypass pre-existing Ad5 immunity inmice [15], macaques [16], and humans [17] probably due tohigh-efficiency gene delivery into cells in the superficial layer alongthe mucosal barrier in conjunction with potent antigen presentationassociated with this immunocompetent interface tissue. The synergybetween primary and booster applications induced by the AdE double-doseregimen (FIGS. 4A and 5) shows that the rapid anti-influenza responsesinduced by AdE were additive in the presence of pre-existing Ad5immunity. These findings hold promise that this nasal influenza DVD notonly is able to induce rapid and sustained protection against influenzain a single-dose regimen but also may be administered repeatedly (e.g.,when a different HA is required for its vaccine component) withoutlosing potency.

Although prophylactic influenza therapy can be performed by i.n.administration of complex bacterial lysates [18] or bacterial toxins[19], the bacterial component-induced anti-influenza state was verytransient with its protective effects declining within a few dayspost-therapy [18,19]. The finding that AdE-induced protective effectscould persist for at least 3 weeks (FIG. 8) and up to 47 days (FIG. 4A)in a single-dose regimen suggests that the underlying mechanisms betweenbacterial component- and Ad5-induced anti-influenza states may differ.In addition, only the latter would allow sufficient time for the DVDsvaccine component to elicit adaptive immunity before its drug effectsdecline away. Moreover, the replicating wild-type Ad5 is a benignrespiratory virus and its non-replicating counterpart used in this studyshould be even safer; notably, the safety profile of an Ad5-vectorednasal influenza vaccine in human subjects has been shown [17]. As acommon respiratory virus, the human mucosal immune system is familiarwith Ad5 particles and must have evolved Ad5-specific protectivemechanisms. In contrast, administration of a digestive tract-associatedbacterial toxin into the respiratory tract as an influenza drug [19]would surprise the immune system and this unnatural regimen has beenassociated with the induction of Bell's palsy in human subjects [20].

The IFV is insidious in mutating into drug-resistant strains when it isattacked by an influenza drug [e.g., the M2 ion channel blocker(amantadine; rimantadine) or the neuraminidase inhibitor (oseltamivir;zanamivir)] [5]. Unlike contemporary influenza drugs, the Ad5-vectoredDVD conceivably changes the habitat in the respiratory tract withoutdirectly attacking the IFV; hence the DVD confers no mutational pressureto induce drug resistance. In contrast to the oseltamivir-inducedsuppression of mucosal immunity with the risk to enhance vulnerabilityto subsequent mucosal pathogen infections [21], the AdS-vectored DVDenhances mucosal innate immunity against at least a subset of mucosalpathogens. The DVD's efficacy is further fortified by its vaccinecomponent that elicits sustained adaptive immunity before its drugeffects completely disappear (FIGS. 4A, 8 and Table 1). Since thelicensed LAIV (e.g., FluMistH in the U.S.) contains live IFV [1],co-administration of LAIV with an influenza drug would becounter-productive because the drug would disable the vaccine by killinglive IFV. The Ad5-vectored DVD not only is compatible with a licensedinfluenza drug, but also it confers prophylactic therapy as a drug byitself in addition to its vaccine capacity.

Emerging evidence shows that a number of nasal vaccines induce a weakersystemic adaptive immune response than their parenteral counterparts[22-26] even though nasal vaccines confer more robust protection againsta respiratory mucosal pathogen by eliciting a more potent mucosaladaptive immune response [22,25]. Applicant provides evidence that notonly adaptive immunity but also innate immunity could be induced with afocus on the respiratory tract against mucosal pathogens when the ΔE1E3Ad5 particle is administered i.n. but not i.m., as shown by % survivalafforded by i.n. and i.m. routes, respectively (FIG. 3). Whether theAd5-vectored nasal DVI) can confer protection against influenza inducedby other routes (e.g., oral infection) remains to be seen.

The finding that i.n. administration of AdNC.H1.1 on day −47 inducedmore robust protection against PR8 challenge than its counterpartinoculated on day −2 or AdE administered on day −47 (FIGS. 3B and 4A)suggests that animals in the AdNC/in/−47 group may be protected by anNC20 HA1-mediated adaptive immune response that cross-reacted with PR847 days post-immunization in the absence of detectable serum HI antibodyto PR8 (Table 1). The data corroborate other reports that serum Hiantibody titer is an inadequate surrogate marker for predictingprotective immunity induced by a nasal influenza vaccine [24,26].

The findings that the Ad5-vectored DVD can confer prophylactic therapyin conjunction with vaccination in a single package provide a foundationfor the development of a novel anti-influenza agent that can bemass-produced in cultured cells, administered painlessly by nasal spray,with the capacity to bypass pre-existing Ad5 immunity and mobilize theinnate as well as the adaptive immune repertoires toward a rapid andsustained beneficial response against influenza, without the potentialto generate drug resistant IFV strains.

Adenovirus. To generate the AdE particle, homologous recombinationbetween the shuttle pAdHigh and the Ads backbone pAdEasy-1 plasmids wasperformed in Escherichia coli BJ5183 cells followed by generation of theRCA-free AdE particle in PER.C6 cells (provided by Crucell Holland BV;Leiden, The Netherlands) as described [1]. AdE is thus a ΔE1E3 Ad5 withan expression cassette in its E1 region [1] without encoding anytransgene. To generate the AdNC.H1.1 vector, the NC20 HA gene wassynthesized at GENEART (Regensburg, Germany) with codons optimized tomatch the tRNA pool found in human cells in conjunction with theinsertion of a eukaryotic ribosomal binding site immediately upstreamfrom the initiation ATG codon [27]. The NC20 HAl fragment containing 347amino acids was amplified from the synthetic HA template by polymerasechain reaction (PCR) using primers 5′-CACAGGTACCGCCACCATGAAGGCCAAGCTG-3′and 5′-GAGTCTAGATTATCAGCCGAACAGGCCTCTGCTCTGG-3′. The KpnI-XbaI fragmentcontaining the amplified HA1 fragment with a stop codon added in-framewas inserted into the KpnI-XbaI site of pAdHigh in the correctorientation under transcriptional control of the human cytomegalovirus(CMV) early promoter. An RCA-free Ad5 vector encoding the NC20 HA1(AdNC.H1.1) was subsequently generated in PER.C6 cells as describedabove. Both AdE and AdNC.H1.1 were validated by DNA sequencing;mass-produced in PER.C6 cells; purified by ultracentrifugation over acesium chloride gradient as described [27]; dialyzed into A195 buffer[28] with titers (ifu per ml) determined in 293 cells [17] by theSpearman-Karber method [29] after staining Ad5-infected monolayers witha horseradish peroxidase (HRP)-conjugated anti-Ad5 hexon antibody andthe 3,3′-diarninobenzidine tetrahydrochloride (DAB) substrate (ClontechLaboratories, Inc.; Mountain View, Calif.). The E1+/E3+ wild-type Ad5(VR-1516) was obtained from the American Type Culture Collection (ATCC;Manassas, Va.).

Influenza virus. PR8 (VR-95) was obtained from the ATCC and grown inMadin Darby Canine Kidney (MDCK) cells in the presence of TPCK-trypsinas described [17] with titers determined by plaque assay [30]. Themouse-adapted CA04 was generated by Natalia A. Ilyushina and provided byElena Govorkova at the St. Jude Children's Research Hospital (MemphisTenn.). The CA04 virus was adapted to replication in the lungs of Balb/cmice by 9 sequential passages through mouse lungs. Virus was plaquepurified in MDCK cells and a virus stock was prepared by growth in10-day-old embryonated chicken eggs and then MDCK cells as described[31] with titers expressed as cell culture infectious doses (CCID50) asdescribed [32]. NC20 was provided by the Center for Disease Control(CDC; Atlanta, Ga.)

Challenge studies. intranasal administration and i.m. injection of 50 μlof Ad5 particles into young (approximately 2 months old) female Balb/cmice were performed as described [27]. Mice were challenged by i.n.instillation of 50 μl of PR8 containing either 1.4×10⁶ plaque-formingunits (pfu) [equivalent to approximately 4×LD₅₀ (50% lethal dose)] or3.5×10⁶ pfu (equivalent to approximately 10×LD50) at University ofAlabama at Birmingham (UAB), as well as 90 μl of CA04 containing 26105CCID50 (equivalent to approximately 3×D₅₀) at Utah State University(USU). All experiments using mice were performed in accordance with theapproval of the Institutional Animal Care and Use Committees at UAB andUSU (UAB Approval ID, #7705; UAB Animal Welfare Assurance Number,A3255-01; USU Approval ID, #552; USU Animal Welfare Assurance Number,A3801-01). Animal facilities at both UAB and USU have been AAALACaccredited.

PR8 titers in lungs post-challenge. AdE particles were i.n. administeredinto young female Balb/c mice at a dose of 1.2×10⁸ ifu in a volume of 50μl on day −2. Five to seven days after i.n. instillation of 4.6×10⁶ pfuof PR8 on day 0, control and AdE-exposed mouse lungs were immediatelyfrozen on dry ice after resection and stored at 280 uC until analysis(FIG. 7). After thawing, a fraction of each lung was weighed andhomogenized in cold phosphate buffered saline (PBS) as a 10% (w/v)suspension. Tissue debris was removed by centrifugation and thesupernatant was transferred to another sterile tube for virus titration.Plaque assay of IFV was performed as described [30].

Hemagglutination-inhibition assay. Sera were tested for activity againstPR8 or NC20 by standard HI assay after pre-treatment of the sera with areceptor-destroying enzyme as described [17]. Each serum sample wastested beginning at a dilution of 1:10. All sera were tested in ablinded fashion on code-labeled, matched pre- and post-immunizationsamples. Animals were considered seronegative and assigned an HIantibody titer of 5 (2.3 on a log 2 scale) if their serum specimen hadan HI titer of <10.

Lung histopathology assay. Mouse lungs were fixed by perfusing 10%buffered. formalin through the trachea. Paraffin-embedded tissues werecut into 5-μm-thick slices followed by staining sections withhematoxylin and eosin (FIG. 6).

Statistical analysis. All statistical analysis was performed usingGraphPad Prism version 5.04 (GraphPad Software, San Diego, Calif.).Log-rank tests were performed for comparing Kaplan-Meier survivalcurves; and one-way ANOVA with Turkey's multiple comparison post-testswere performed for comparing body weight loss as well as PR8 titers inlungs. Statistical significance was set at P,0.05.

REFERENCES

-   1. Tang D C, Zhang J, Toro H, Shi Z, Van Kampen K R (2009)    Adenovirus as a carrier for the development of influenza virus-free    avian influenza vaccines. Expert Rev Vaccines 8: 469-481.-   2. Neumann G, Watanabe T, Ito H, Watanabe S, Goto H, et al. (1999)    Generation of influenza A viruses entirely from cloned cDNAs. Proc.    Natl. Acad. Sci. USA 96: 9345-9350.-   3.Wei C J, Boyington J C, McTamney P M, Kong W P, Pearce M B, et    al. (2010) Induction of broadly neutralizing H1N1 influenza    antibodies by vaccination. Science 329: 1060-1064. 4. 4.

Konig R, Steitz S, Zhou Y, Inoue A, Hoffmann H H, et al. (2010) Humanhost factors required for influenza virus replication. Nature 463:813-817.

-   5. Poland G A, Jacobson R M, Ovsyannikova I G (2009) influenza virus    resistance to antiviral agents: a plea for rational use. Clin Infect    Dis 48: 1254-1256.-   6. Hartman Z C, Appledore D M, Amalfitano A (2008) Adenovirus vector    induced innate immune responses: impact upon efficacy and toxicity    in gene therapy and vaccine applications. Virus Res 132: 1-14.-   7. Yamaguchi T, Kawabata K, Kouyama E, Ishii K J, Katayama K, et    al. (2010) Induction of type I interferon by adenovirus-encoded    small RNAs. Proc Nati Acad Sci USA 107: 17286-17291.-   8. Thiele A T, Sumpter T L, Walker J A, Xu Q, Chang C H, et    al. (2006) Pulmonary immunity to viral infection: adenovirus    infection of lung dendritic cells renders T cells nonresponsive to    interleukin-2. J Virol. 80: 1826-1836.-   9. Zhu J, Huang X, Yang Y (2008) A critical role for type I    IFN-dependent NK cell activation in innate immune elimination of    adenoviral vectors in vivo. Mol Ther 16: 1300-1307.-   10. Higashimoto Y, Yamagata Y, Itoh H (2006) Complex effect of    adenovirus early region proteins on innate immune system. Inflamm    Allergy Drug Targets 5: 229-237.-   11. Schaack J, Bennett M L, Colbert J D, Torres A V, Clayton G H, et    al. (2004) E1A and E1B proteins inhibit inflammation induced by    adenovirus, Proc Natl Acad Sci USA 101: 3124-3129.-   12. Rhee E G, Blattman J N, Kasturi S P, Kelley R P, Kaufman D R, et    al. (2011) Multiple innate immune pathways contribute to the    immunogenicity of recombinant adenovirus vaccine vectors. J Virol    85: 315-323.-   13. Goodman A G, Zeng H, Proll S C, Peng X, C, et al. (2010) The    alpha/beta interferon receptor provides protection against influenza    virus replication but is dispensable for inflammatory response    signaling. J Virol 84: 2027-2037.-   14. Tumpey T M, Szretter K J, Van Hoeven N, Katz J M, Kochs G, et    al. (2007) The M×1 gene protects mice against the pandemic 1918 and    highly lethal human H5N1 influenza viruses. J Virol 81: 10818-10821.-   15. Croyle M A, Patel A, Tran K N, Gray M, Zhang Y, et al. (2008)    Nasal delivery of an adenovirus-based vaccine bypasses pre-existing    immunity to the vaccine carrier and improves the immune response in    mice. PLoS ONE 3: e3548.-   16. Song K, Bolton D L, Wilson R L, Camp J V, Bao S, et al. (2010)    Genetic immunization in the lung induces potent local and systemic    immune responses. Proc Natl Acad Sci USA 107: 22213-22218.-   17. Van Kampen K R, Shi Z, Gao P, Zhang J, Foster K W, et al. (2005)    Safety and immunogenicity of adenovirus-vectored nasal and    epicutaneous influenza vaccines in humans. Vaccine 23: 1029-1036.-   18. Tuvim M J, Evans S E, Clement C G, Dickey B F, Gilbert B    E (2009) Augmented lung inflammation protects against influenza A    pneumonia. PLoS ONE 4: e4176.-   19. Norton E B, Clements J D, Voss T G, Ca'rdenas-Freytag L (2010)    Prophylactic administration of bacterially derived immunomodulators,    improves the outcome of influenza virus infection in a murine    model, J. Virol. 84: 2983-2995.-   20. Couch R B (2004) Nasal vaccination, Escherichia coli    enterotoxin, and Bell's palsy. N. Engl. J. Med 350: 860-861.-   21. Takahashi E, Kataoka K, Fujii K, Chida J, Mizuno D, et    al. (2010) Attenuation of inducible respiratory immune responses by    oseltamivir treatment in mice infected with influenza. A virus,    Microbes infect 12: 778-783.-   22. Wang J, Thorson L, Stokes R W, Santosuosso M, Huygen K, et    al. (2004) Single mucosal, but not parenteral, immunization with    recombinant adenoviral-based vaccine provides potent protection from    pulmonary tuberculosis. J Immunol 173: 6357-6365.-   23. Lemiale F, Kong W P, Akyurek L M, Ling X, Huang Y, et al. (2003)    Enhanced mucosal immunoglobulin A response of intranasal adenoviral    vector human immunodeficiency virus vaccine and localization in the    central nervous system. J Virol 77: 10078-10087.-   24. Hoelscher M A, Garg S, Bangari D S, Belser J A, Lu X, et    al, (2006) Development of adenoviral-vector-based pandemic influenza    vaccine against antigenically distinct human H5N1 strains in mice.    Lancet 367: 475-481.-   25. Price G E, Soboleski M R, Lo C Y, Misplon J A, Quirion M R, et    al. (2010) Single dose mucosal immunization with a candidate    universal influenza vaccine provides rapid protection from virulent    H5N1, H3N2 and H1N1 viruses. PLoS ONE 5: e13162.-   26. Clements M L, Betts R F, Tierney E L, Murphy B R (1986) Serum    and nasal wash antibodies associated with resistance to experimental    challenge with influenza A wild-type virus. J Clin Microbiol 24:    157-160.-   27. Shi Z, Zeng M, Yang G, Siegel F, Cain L J, et al. (2001)    Protection against tetanus by needle-free inoculation of    adenovirus-vectored nasal and epicutaneous vaccines. J Virol 75:    11474-11482.-   28. Evans R K, Nawrocki D K, Isopi L A, Williams D M, Casimiro D R,    et al. (2004) Development of stable liquid formulations for    adenovirus-based vaccines. i Pharm Sci 93: 2458-2475.-   29. Lynn D E (2001) Effects of temperature on the susceptibility of    insect cells to infection by baculoviruses. Methods Cell Sci 23:    221-225. 30. Gaush C R, Smith T F (1968) Replication and plaque    assay of influenza virus in an established line of canine kidney    cells. Appl Microbiol 16: 588-594.-   31. Ilyushina. N A, Khalerikov A M, Seiler J P, Forrest H L, Bovin N    V, et al. (2010) Adaptation of pandemic H1N1 influenza viruses in    mice. J Virol 84: 8607-8616.-   32. Barnard D L, Wong M H, Bailey K, Day C W, Sidwell R W, et    al. (2007) Effect of oral gavage treatment with ZnAL42 and other    metal to-ion formulations on influenza A H5N1. and H1N1 virus    infections in mice. Antivir Chem Chemother 18: 125-132.

Example 3 Adenovirus-Vectored Drug-Vaccine Duo as a Potential Driver forConferring Mass Protection Against Infectious Diseases

The disease-fighting power of vaccines has been a public health bonanzacredited with the worldwide reduction of mortality and morbidity. Thegoal to further amplify its power by boosting vaccine coverage requiresthe development of a new generation of rapid-response vaccines that canbe mass produced at low costs and mass administered by nonmedicalpersonnel. The new vaccines also have to be endowed with a higher safetymargin than that of conventional vaccines. The nonreplicatingadenovirus-vectored vaccine holds promise in boosting vaccine coveragebecause the vector can be rapidly manufactured in serum-free suspensioncells in response to a surge in demand, and noninvasively administeredby nasal spray into human subjects in compliance with evolutionarymedicine. In contrast to parenteral injection, noninvasive mucosalvaccination minimizes systemic inflammation. Moreover, preexistingadenovirus immunity does not interfere appreciably with the potency ofan adenovirus vectored nasal vaccine. Nasal administration of adenovirusvectors encoding pathogen antigens is not only fear-free and painless,but also confers rapid and sustained protection against mucosalpathogens as a drug-vaccine duo since adenovirus particles alone withouttransgene expression can induce an anti-influenza state in the airway.In addition to human vaccination, animals can also be mass immunized bythis class of vectored vaccines.

A litany of demands for better vaccines. Although vaccination proves tobe the most cost-effective method for the prevention of disease, asweeping offensive to boost vaccine coverage remains a compelling goalin the movement toward improved public health worldwide. Currentvaccines that have been licensed for marketing include killed wholemicroorganisms, live attenuated microorganisms, microbial extracts,purified or recombinant proteins, DNA vaccines and virus-like particles.Even though many diseases have been defeated by the broad distributionof these vaccines, the goal to generate community (herd) immunity in awide variety of disease settings remains elusive owing to a number ofproblems in current vaccination programs. Specifically,vaccine-associated adverse side effects range from local and systemicinflammatory response, fever, platelet activation, cardiac autonomicdysfunction, anaphylactic reaction (induced by needle injection ofcertain vaccines) [1-4] to the rare occurrence of paralyticpoliomyelitis (mediated by ingestion of the oral polio vaccine) [5],myopericarditis (induced by inoculation of the Dryvax smallpox vaccine)[6] and Bell's palsy (induced by a bacterial toxin nasal adjuvant)[7,8]. In 2010, a sudden rise of narcolepsy among vaccinees was reportedin a few countries following needle injection of an H1N1 pandemicinfluenza vaccine containing the squalene adjuvant [201]. Injection ofsqualene alone can induce rheumatoid arthritis in animals [9]. Asemerging evidence shows that chronic, low-grade inflammation isassociated with cardiovascular disease [10], obesity [11], diabetes[11], cancer [12] and neurological disorder [13], vaccine-inducedinflammation now needs focused attention. Whether an acute inflammatoryreaction induced by injection of an immunostimulating vaccine-adjuvantcomplex [1-3] could evolve into a chronic, tow-grade inflammation andtrigger any of these ailments in a subset of vaccinees over time is ofparamount importance in public health; however, this potential hazardhas not been rigorously investigated. Since the concept of vaccinesafety is evolving from ‘protection against pathogen-induced diseases’to ‘no possibility of inducing adverse consequences’, any knownextraneous agents, toxicity and residual virulence found in a vaccinewould not be allowed, and any possibility of inducing unknown sideeffects (e.g., inflammation in vital organs) should be avoided.

Mucosal and systemic immune responses are elicited and regulated with aconsiderable degree of independence and most vaccines have beenadministered invasively by intramuscular injection, which induces goodsystemic immunity but often weak mucosal immunity that is crucial indefense against mucosal pathogens (e.g., influenza virus, Mycobacteriumtuberculosis and HIV) [14,15]. Efficient induction of mucosal immunityusually employs nasal or oral vaccination owing to the unique ability ofresident mucosal dendritic cells (DCs) to induce IgA switching and toimprint mucosa-specific homing receptors (e.g., CCR9 and a4b7 integrin)on lymphocytes [15,16]. In addition to weak mucosal immunity induced byan injectable vaccine, the syringe needle as a vaccine administrationdevice also poses serious problems through intentional or inadvertentunsterile re-use, needlestick injury, improper waste disposal, as wellas limited injection service by licensed medical personnel during acrisis [17]. Public fear of pointed needles (aichmophobia) plays anotherrole in hindering vaccine coverage. Some people may thus prefer the oddsof getting a disease versus the odds of inflicting pain, injury, ordeath by systemic vaccination. Since the objective of vaccinationprograms is to reduce the overall probability of infection by generatingcommunity (herd) immunity, the mission will be undermined by a hold-offon vaccination owing to public fear of risks. To date, enablingtechnologies for reversing negative perceptions by developing a newgeneration of rapid-response vaccines that are safe, efficacious,painless and economical are emerging on the horizon.

Noninvasive vaccination as a means to boost vaccine coverage.Needle-free noninvasive vaccination holds the promise of changing thepublic's attitude from ‘being forced to get a needle shot’ to‘proactively seeking vaccination without fear’. Vaccines can beadministered noninvasively by oral ingestion [5], nasal spray [18,19],as well as topical application of a skin patch [19-23] in a painlessmanner. Noninvasive vaccination by administration of vaccines to theinterface between the inner body and outside environment not onlyconfers a high degree of vaccinee comfort, but may also lead to aqualitatively superior immune response as compared with conventionalsystemic vaccination. Mucocutaneous surfaces are covered by a highlyimmunocompetent epithelium that serves as a physical barrier and ensuresthat antigens penetrating into the superficial layer are efficientlycaptured and presented to the immune system. By logic, animals andhumans must deploy the most competent immune cells along the surfacebarrier to ward off infections since it would be counter-productive tokeep these ‘professional immune soldiers’ in deep tissues where theyrarely encounter invading pathogens. Professional antigen-presentingcells (APCs) including multiple DC subsets [24,25], gdT cells [26] andothers can be found in high densities along the mucocutaneous surface. Asubset of mouse bone marrow cells expressing the retinoicacid-synthesizing enzyme are capable of providing retinoic acid to DCprecursors for inducing mucosal DC functions, including generation ofFoxp3+ regulatory T cells, IgA-secreting B cells and mucosa-specifichoming receptors [27]. It has been shown that the route of vaccinationcritically impacts not only the magnitude but also the phenotype andtrafficking of antigen-specific CD8+ T lymphocytes in mice.Intramuscular injection of an adenovirus (Ad)-vectored vaccine inducedrobust local transgene expression and elicited high-frequency,polyfunctional CD8+ T lymphocytes that trafficked broadly to bothsystemic and mucosal compartments. By contrast, intranasal instillationof the Ad-vectored vaccine led to similarly robust local transgeneexpression but generated low-frequency, monofunctional CD8+ Tlymphocytes with restricted anatomic trafficking patterns [28].Noninvasive vaccination thus takes advantage of an existing biologicalpathway that leverages the immune system's ability to respond atsuperficial but immunocompetent tissue sites along the mucocutaneoussurface to elicit localized protective immunity against mucosalpathogens at the portal without inducing an over-reactive systemicimmune response.

Although it is required to aseptically manufacture vaccines undercurrent good manufacturing practices, contamination by unknownmicroorganisms or contamination below detection by modern instrumentsand high-throughput assays may still occur. Coadministration of thesecontaminants with a noninvasive vaccine onto the mucocutaneous surfacewould pose little danger to the vaccinee since the mucocutaneous immunesystem is well versed in counteracting microbial invasion at all timesas the interface is in constant contact with microbes. By contrast,injection of a contaminated vaccine into deep tissues can, in theory,trigger an exponential growth of microorganisms within the body in theabsence of a timely immune response, or conversely, an ‘immune storm’induced by an over-reacting immune system. Overall, elicitation ofprotective immunity along the mucocutaneous surface is a daily routine;animals and humans have evolved adequate mechanisms for winning dailybattles (daily microbial invasion) without losing the war (overallhealth). Noninvasive vaccination utilizes the daily operation of theimmune system along the interface without surprising the immune systemby physical delivery of immunostimulating vaccine-adjuvant complexesinto deep tissues where immunocompetence is low.

The zigzag pathway to develop an adenovirus into a vaccine carrier.Adenovirus belongs to a family of icosahedral nonenveloped DNA viruseswith a linear DNA genome of 30-38 kb (size varies from group to group)bracketed by inverted terminal repeats. An Ad particle contains atightly coiled DNA genome packaged inside a hexagonal protein capsid(FIG. 9). The Ad genome contains both early genes encoding regulatoryproteins and late genes encoding structural proteins [29]. Multiple Adserotypes are commonly found in animals and humans, and there can besignificant differences in the pathogenicity and course of disease amongdifferent serotypes; some are quite benign in immunocompetent humanhosts (e.g., human Ad serotype 5 [30]) whereas others may cause diseasesthat are usually mild and self-limiting. A number of noteworthy reasonswarrant the development of Ad into a vaccine carrier. Specifically,human Ad4- and Ad7-based oral vaccines (a type of noninvasive vaccine)have proven safe as well as efficacious during mass immunization ofmilitary recruits [31]. Potentially, replicating Ad4 or Ad7 can hefurther bioengineered into oral vaccine carriers to elicit immunityagainst other pathogen-derived antigens. However, it is difficult toquantitatively release a replicating bioengineered vector thatrepresents a genetically modified organism in a controlled manner,Introduction of a genetically modified organism into the ecosystem isalso undesirable in the public's perception. A nonreplicating vectorthus would be safer and more acceptable than its replicatingcounterpart. Although a nonreplicating Ad5 vector was developed nearlythree decades ago by truncating its E1 region (FIG. 9) [32], a criticalissue for the E1-defective (ΔE1) Ad5 vector produced in human 293 cellsis the intrinsic contamination by replication-competent Ad (RCA) thatarises through homologous recombination between overlapping sequencesframing the E1 locus displayed by transfected 293 cells and the vectorbackbone [33], RCA represents a biohazard because it can replicate in aninfected host with the capacity for horizontal transmission tobystanders through virus shedding [30]. To circumvent the problem ofRCA, RCA-free Ad vectors have been generated in human PER.C6 cells usingPER.C6-compatible shuttle vectors that do not contain overlappingsequences with the PER.C6 genorne [34,35]. Unlike replicating Ad4 andAd7, the nonreplicating Ad5 does not immunize animals efficiently whenadministered orally due to its inability to undergo virus amplificationand its susceptibility to low pH, gastric and pancreatic proteases, andextracellular mucins [36].

Despite problems observed following administration by the oral route,the nonreplicating E1/E3-defective (AEIE3) Ad5 (FIG. 9) has beendeveloped and used as a parenteral gene-therapy vector in a large numberof therapeutic trials owing to its high capacity for accommodatingtransgenes, high-titer production, high-efficiency gene delivery andhigh-level transgene expression (at least as an initial burst) [35].However, Ad5 is not an ideal vector for classical gene therapy becausetransgene expression is transient [35]. Therefore, use of an Ad5 vectorfails to meet a major goal of gene therapy, which usually requiressustained transgene expression. Moreover, the common presence ofpre-existing Ad5 immunity in human populations [37-39] and the rapiddevelopment of an anti-Ad5 immune response following the firstinoculation with the vector [40, 41] have impeded its clinical use bylimiting gene transfer efficiencies. Strategies to circumvent thesedrawbacks include serotype switching, capsid modification anddevelopment of nonhuman Ad vectors, on the assumption that another Advector can :substitute when the initial one is disabled by pre-existingAd immunity. Although human Ad3, Ad4, Ad35, Ad41 or a chimeric Adscontaining the Ad11 or Ad35 fibers have been bioengineered intononreplicating Ad vectors (FIG. 10), Ad5 is still more potent and saferthan other serotypes in preclinical animal models [42]. A number ofnonhuman Ads such as bovine Ad [43], porcine Ad [43] and nonhumanprimate Ad [44] have also been developed to expand the repertoire of Advectors (FIG. 10). Even though a porcine Ad-vectored vaccine can be atleast as potent as its Ads counterpart in mice [45], the human Ad5 isstill the premier gene-transfer vector owing to the risk of inducingunpredictable human ailments by a nonhuman Ad [46]. The genome of humanAd5 is remarkably stable in the field even after coinfection with otherAd serotypes [47]. Moreover, the Ad5 vector has been further developedto display foreign antigens on the surface after fusing pathogenepitopes to the pIX [48] or the hexon capsid proteins [49] in additionto encoding pathogen antigens in its DNA genome (FIG. 10). Lessimmunogenic Ad5 vectors were developed by deletion of E2b [50, 51] ornearly all Ad5 sequences except the inverted terminal repeats and thepackaging signal (gutless Ad) (FIG. 10) [52]. To date, thesesophisticated strategies have not yet yielded profound clinicalimprovement.

In contrast to intramuscular or intravenous injection of Ad5, it hasbeen shown that intranasal administration (the natural route of Ad5infection) would allow a ΔE1E3 Ad5-vectored vaccine to bypasspre-existing Ad5 immunity without appreciably losing potency in mice[41, 53], nonhuman primates [5.4] and humans [19]. These observationsare conceivably attributed to the high efficiency of gene delivery,robust transgene expression and potent antigen presentation along themucosal barrier in the respiratory tract. Anti-Ad5 immunity is thus nolonger an insurmountable limiting factor and refinement of bioengineeredAd vectors may no longer be a sine qua non for further development ofAd-vectored vaccines.

The Ad5 vector's reputation has been derailed multiple times during itsdevelopment. In addition to pre-existing Ad5 immunity, the death of apatient with partial ornithine transcarbamylase (OTC) deficiency afterinfusing a high dose of Ad5-OTC vector into his hepatic artery during ahuman gene-therapy trial [55] marked Ad5 as a dangerous vector in thepublic's perception. Evidence shows that injection of Ad particles intothe circulatory system (an unnatural route for Ad infection) is anunsafe approach because Ad particles rapidly induce systemicinflammation postinjection [56, 57], and a variety of Ad serotypes causeactivation of coagulation, possibly through interaction with platelets[42]. During a large-scale human trial (Step Study) of an Ad5-vectoredHIV vaccine, administration by intramuscular injection did not lower HIVviral load and vaccination was associated with increased risk of humanHIV infection in Ad5-seropositive subjects [58, 59]. Thecounterintuitive results may again be attributed to a misuse of thevector since the potency of an Ad5-vectored vaccine surpasses that ofother virus- and nonvirus-based vaccine platforms in eliciting cellularimmunity [60]; consequently, the Ad5-induced expansion of CD4+ T cellswould exacerbate this peculiar disease as CD4+ T cells are the specifictargets for HIV infection [61]. In addition, human subjects wereimmunized by intramuscular injection of Ad5 particles during the StepStudy [58, 59], which is not very potent in eliciting mucosal immunityagainst a mucosal pathogen such as HIV [14,62,63].

Reality check of current adenovirus-vectored vaccines. To develop thenext generation of vaccines that are safe and effective, it is crucialfor the vaccine to induce protective immunity rapidly with a highbenefit-to-risk ratio. The manufacture, distribution and administrationof the vaccine must be easy, fast and economical. In addition, theinherent stability of the formulated vaccine and final filled producthas to allow for long-term stockpiling without a cold chain.

As shown in Table 2, protective immunity against a wide variety ofpathogens has been elicited in mice, guinea pigs, chickens, hamsters,cotton rats, raccoons, skunks, pigs and nonhuman primates followingimmunization with Ad-vectored vaccines. Overall, Ad-vectored vaccinescan confer rapid and more robust protection against live pathogens thanother types of vaccines in animal models.

Although multiple human clinical trials of Ad-vectored vaccines havebeen performed, few immunized human subjects have been challenged with avirulent live pathogen (Table 3). Notably, a subset of human volunteersimmunized by intramuscular injection of DNA and Ad5-vectored malariavaccines (DNA-primed/Ad5-boosted) were protected against live malariasporozoite challenge following mosquito feeding in Ad5-seronegativehuman subjects. It has been shown that DNA vaccination alone without Ad5booster failed to protect humans against malaria; whether Ad5vaccination alone could confer protection remains to be seen [64]. Eventhough immunized humans were not challenged with live pathogens duringmost human trials (Table 3), they have provided a broad safety databasefor the use of Ad vectors in humans.

Potency & safety of adenovirus-vectored nasal vaccines. As describedearlier, nasal vaccination induces potent mucosal immunity in aneedle-free manner. Respiratory tract DCs form a contiguoussubepithelial network within the nasorespiratory tract, bridging innateand acquired immunity. The density of DCs within the respiratory tractis highest in those areas exposed to greater amounts of inhaled antigen[65]. Nasopharynx-associated lymphoid tissue, constituting Waldeyer'sring in humans, is a unique inductive site for B-cell responses andplasma cell generation. Nasal vaccination is thus a driver for theelicitation of humoral immunity including the formation of secretory IgAantibody within the respiratory tract [66]. Local humoral immuneresponses have been induced in nasal, vaginal and salivary secretionsfollowing intranasal administration of Ad-vectored vaccines intononhuman primates [67]. An Ad5-vectored nasal vaccine induced greaterantigen-specific IgA responses in mucosal secretions and sera in micethan its injectable counterpart [68]. In addition to humoral immunity,cellular immune responses were observed in systemic and mucosal immunecompartments shortly after immunizing mice with an Ad-vectored herpesvaccine regardless of the route of inoculation; however, anamnesticcytotoxic T lymphocyte responses compartmentalized exclusively tomucosal or systemic lymphoid tissues after mucosal or systemicimmunization, respectively, several months postimmunization [14].

Although the DNA-primed/Ad5-boosted malaria vaccine induced protectionagainst live malaria sporozoite challenge in Ad5-seronegative humansubjects, the failure to protect five Ad5-seropositive human volunteers[64] may be attributed to pre-existing Ad5 immunity [37, 38, 40, 41]. Asdescribed earlier, one approach to circumvent this hurdle is toinoculate Ad-vectored vaccines by nasal administration, which leverageswhat is a disadvantage for injectable vaccines to an advantage fornoninvasive mucosal vaccines without reduced effectiveness of subsequentAd5 re-administration [19, 41, 53, 54]. An Ad5-vectored nasal vaccinemay induce focused mucosal immunity in the airway, as shown by findingsthat intranasal immunization, but not systemic immunization, induceslong-lived cytotoxic T lymphocytes in mucosal tissues [14]. In addition,Ad5-vectored nasal vaccines can protect animals against mucosalpathogens when systemic immunization fails, even though the latterinduces a more robust systemic immune response [63, 69, 70]. Thehypothesis that the focused mucosal immune response induced by nasalvaccination may greatly reduce the systemic burden (e.g., systemicinflammation) to unaffected internal tissues and organs was borne out bythe finding that CD 103+ mucosal DCs can dampen inflammatory responsesby fostering the conversion of naive T cells into Foxp3⁺ regulatory Tcells [71].

The common adverse effects induced by systemically delivered Adparticles are liver damage and systemic toxicity owing to sequestrationof Ad particles to the liver in large numbers following injection [72].In contrast to parenteral injection, biodistribution of Ad is limited tothe lung following intranasal administration [73] with no inflammationobserved in any of the internal organs [68].

Owing to the proximity of the nasal cavity to the brain; it is crucialto determine whether Ad5 particles may induce inflammation and toxicityin the brain following nasal spray. Unlike influenza, which isassociated with human neurological disorders [74], natural infection byAd5 has not been reported to induce encephalitis in humans. Intranasaladministration of ΔE1E3 Ad5 vectors into mice did not mediate transgeneexpression beyond the olfactory bulb, nor induction of inflammation inthe brain [68]. It is thus conceivable that significant amounts of Ad5cannot enter the brain following nasal delivery, Even though a smallnumber of Ad5 particles may infiltrate into the brain on occasion, thenonreplicating Ad5 is likely to do less harm than its replicatingwild-type counterpart due to its inability to amplify adverse effectsthrough replication and late gene expression. The safety profile of thelive-attenuated influenza virus vaccine (LAIV; known as HuMist® in theUSA) [75] corroborates the hypothesis that the influenza virus-inducedencephalitis [74] could be attributed to viral replication in the brainsince LAIV can only replicate in the airway, where temperature is lower,but not within the brain, where it is too hot for the cold-adapted LAIV.The induction of herpes simplex encephalitis in TIR-3-deficient patients[76] suggests that it may be a common event fur a small amount of virusto penetrate the brain through the olfactory tract and that an effectivedefense mechanism exists in immunocompetent people to arrest the virusbefore it replicates uncontrollably within the brain. Since naturalinfection by replicating wild-type Ad5 is not associated withencephalitis, nasal spray of nonreplicating Ad5 vector thus represents adriver in the pursuit of a safe carrier for vaccine delivery.

Even though it may have been a mistake to immunize humans viaintramuscular injection of an AdS-vectored HIV vaccine [58, 59], theability of Ad5 to mobilize the CD4+ T-cell repertoire may be the driver,in part, for eliciting potent protective immunity against otherpathogens [41, 53, 63, 77-79]. To date, intranasal administration of anAd5-vectored influenza vaccine has induced seroconversion in humansubjects without causing serious side effects in the presence ofpre-existing Ad5 immunity [19]. The induction of sterile immunityagainst malaria [64] and seroconversion against influenza [19] in humans(Table 2) in conjunction with solid protective immunity induced inmultiple animal models (Table 2) collectively prove the worth ofAd-vectored vaccines in preventing disease.

LAIV has been licensed for immunizing a subset of human populations(2-49 years of age in the USA) [75]. Like LAIV, it is conceivable thatan Ad-vectored nasal vaccine may not be permitted to immunize the veryyoung and the elderly, at least during the initial period before itssafety profile is well established through large-scale field trials.Furthermore, nasal vaccination would not be recommended for people withrespiratory illness (e.g., asthma) Whether pregnant women will beamenable to nasal vaccination using nonreplicating Ad particles remainsto be seen.

Prospect for commercialization of Ad-vectored vaccines & otherrecombinant DNA-based vaccines. The nonreplicating ΔE1E3 Ad5-vectoredvaccine without RCA contamination [35] can be classified as a variant ofDNA vaccines because it consists of a linear DNA genome embedded in aprotein capsid (FIG. 9) without the capability of replication innonpermissive cells. Unlike naked DNA vaccines that have to beinoculated by trained personnel using a penetrating device such as thegene gun [80,81], syringe needle [82] or electroporator [83], Adparticles can autonomously penetrate cells along the mucosal barrierfollowing nasal delivery [35]. Only a decade ago, DNA vaccines were anunproven novelty with limited acceptance in the scientific community,even though DNA vaccines forego many of the potential safety concernsrelated to contemporary vaccines and recombinant DNA technology cangenerate new vaccines rapidly and creatively at low costs [80, 82]. Todate, four naked DNA vaccines have been licensed for animal use on acommercial scale [84]. An RCA-contaminated. Ad5 vector encoding p53produced in 293 cells has been licensed for treating a large number ofcancer patients in China since 2004 [85]. As the clinical picture isbeginning to unfold as a result of years of increased usage and carefulpatient follow-up, it is conceivable that promising data may usher in arecombinant DNA-based vaccine age with the Ad-vectored vaccine as one ofthe essential tools in the public health arsenal against infectiousdisease.

Maintenance of Ad vector viability during storage. In addition to safetyand efficacy, the next generation of vaccines has to be less reliant ona chain of cold facilities to ensure wide dissemination of vaccines tothe world's least affluent populations. To date, novel formulations haveallowed Ad vectors to be stored in liquid buffer at 4° C. for at least ayear [86]; at 45° C. in carbohydrate glass for at least 6 months [87];or at 4° C. for at least a year as lyophilized dry powder [88].Proprietary technologies for storing Ad particles at room temperature ineither liquid or lyophilized form have also been developed atStabilitech [202]. In summary, RCA-free. Ad5 vectors can be rapidlymanufactured in serum-free PER.C6 suspension cells, purified easily bycolumn chromatography, and formulated as final filled products that canbe stored and shipped without a cold chain (FIG. 11).

Adenovirus-vectored drug-vaccine duo for conferring rapid & sustained,seamless protection against pathogens. Applicant recently demonstratedthat intranasal administration but not intramuscular injection of ΔE1E3Ad5 particles, with or without a pathogen antigen encoded in the Ad5genome, can confer prophylactic therapy against influenza beforeadaptive immunity is elicited [89]. An Ad5 vector encoding pathogenantigens may thus induce rapid and sustained seamless protection againsta pathogen as a drug-vaccine duo (DVD). An Ad5-vectored influenza DVDconfers a number of advantages when it is compared with licensedinfluenza vaccines (Table 4) and drugs (Table 5). It has been documentedthat administration of ΔE1E3 Ad5 particles into mice rapidly induces theproduction of a wide array of inflammatory cytokines and chemokines [56]including type I interferon (IFN-α and IFN-β) [90], impairs lung DCs[91], activates natural killer cells [92], induces production ofantiviral nitric oxide [57], and triggers multifaceted interactionsbetween Ad5 and blood proteins, platelets, macrophages, endothelialcells and respective parenchymal cells [56]. It is conceivable thatmultiple reactions induced by the ΔE1E3 Ad5 particles may combine toestablish an anti-influenza state in the airway, thus creating amultidimensional defense barrier that cannot easily be bypassed by aninfluenza virus.

Although prophylactic influenza therapy can be performed by intranasaladministration of complex bacterial lysates [93] or bacterial toxins[94], the bacterial component-induced anti-influenza state was verytransient, with its protective effects declining within a few dayspost-therapy [93,94], The finding that Ad5-induced protective effectscould persist for at least 3 weeks with only a partial decline observedon day 47 in a single-dose regimen [89] suggests that the underlyingmechanisms between bacterial component- and ΔE1E3-Ad5-inducedanti-influenza states may differ. Notably, only the Ad5-mediated therapywould allow sufficient time for the DVD's vaccine component to elicitadaptive immunity before its drug effects decline (FIG. 12).Furthermore, administration of a digestive tract-associated bacterialtoxin into the respiratory tract as an influenza drug [94] violates acore principle in evolutionary medicine by surprising the immune system,and this unnatural regimen has been associated with the induction ofBell's palsy in human subjects [7,8].

Influenza virus is insidious in mutating into drug-resistant strainswhen it is inhibited by an influenza drug (e.g., the M2 ion channelblocker [amantadine, rimantadine] or the neuraminidase inhibitor[oseltamivir, zanamivir]) [95]. Unlike contemporary influenza drugs, theAd5-vectored DVD conceivably changes the habitat in the respiratorytract without directly effecting the influenza virus; hence the Ad5particle confers no mutational pressure on influenza virus for inducingdrug resistance. In contrast to the oseltamivir-induced suppression ofmucosal immunity with the risk to enhance vulnerability to subsequentmucosal pathogen infections in drugged animals [96], the Ad5-vectoredDVD enhances protective mucosal innate immunity, at least in theinfluenza setting [89]. Since the licensed nasal LAIV (e.g., FluMist)contains live influenza virus [35], coadministration of LAIV with aninfluenza drug is counter-productive because the drug would disable thevaccine by killing live influenza viruses. The Ad5-vectored DVD is notonly compatible with a licensed influenza drug due to its lack of thedrug targets (e.g., ion channel or neuraminidase) (Table 4), but it alsoconfers prophylactic therapy as a drug by itself in addition to itsvaccine capacity (Table 5) [89].

It is unlikely that influenza is the only disease that can be arrestedby Ad5 particles; it is also unlikely that Ad5 can counteract alldiseases as a panacea. The findings merely show that a single intranasaladministration of Ad5-vectored DVD can confer prophylactic therapyagainst at least a subset of mucosal respiratory pathogens for manyweeks in a preclinical animal model and use of the DVD should be unableto induce drug resistance. Subsequent elicitation of sustainedprotective immunity by the DVD's vaccine component fortifies efficacy.The development of a DVD platform will conceivably foster thedevelopment of novel clinical strategies in a wide variety of diseasesettings.

Mass immunization of animals with adenovirus-vectored vaccines. As shownin Table 2, Ad-vectored vaccines have been developed to mass immunizefarm animals as well as wildlife. Notably, chickens can be immunizedagainst avian influenza (possibly other poultry diseases as well) byintramuscular injection [78], in ovo administration [97-101], or aerosolspray [102,103] of human Ad5 vectors encoding avian influenza virushemagglutinin. The versatility of Ad5-vectored vaccines in massimmunization of poultry thus outperforms that of other poultry vaccines.Pigs have also been successfully immunized with Ad5-vectored vaccines[104,105]. An oral canine Ad-vectored rabies vaccine has been developedas a bait to mass immunize wildlife [106]. Overall, Ad-vectored vaccinesare emerging as a promising tool in mass immunization programs.

Conclusion. Evidence shows that Ad-vectored vaccines and the new M)provide a potentially revolutionary approach, allowing preciselydesigned, easily manufactured and highly effective DVD to confer rapidand sustained, seamless protection of humans and animals in a widevariety of disease settings, without the side-effect profile, shelfinstability or manufacturing challenges that other approaches have seen.

Expert commentary. To further boost vaccine coverage worldwide, it isurgent to develop a new generation of vaccines that can be rapidlymanufactured at low costs and mass administered by nonmedical personnelwithout the requirement for a cold chain. Ad5-vectored vaccines complywith these criteria. The development of a DVD platform may potentiallychange the medical landscape by consolidating vaccines and drugs into asingle package that is not impaired by drug resistance.

Five-year view. Two human Phase I clinical trials of Ad5-vectored nasalinfluenza vaccines have been completed with promising results. Challengeof human subjects with live influenza viruses following nasal spray ofan Ad5-vectored. DVD is expected to be performed within 5 years.Ad5-vectored poultry vaccines are expected to enter the commercialmarket within 5 years.

Key issues. There is an urgent need to develop a new generation ofvaccines that can be rapidly manufactured and mass administered bynonmedical personnel during a crisis. Replication-competent adenovirus(RCA)-free adenovirus (Ad)5-vectored vaccines can be produced rapidly atlow costs from PER.C6 suspension cells in serum-free medium in responseto an escalation in demand. RCA-free Ad5-vectored vaccines can be massadministered to people by nasal spray, as well as to poultry byautomated in ovo administration and aerosol spray. Wildlife can be massimmunized by baits containing canine Ad-vectored oral vaccines.Ad-vectored vaccines can induce highly specific immune interventionsbased on well-defined antigens that are the focus of specific immunereactivity. There should not be any safety concerns for nasaladministration of an RCA-free Ad5 vector into people since the vector isnonreplicating and the procedure is in compliance with evolutionarymedicine. There should not be any safety concerns for mass immunizationof poultry by an Ad5 vector since chicken cells do not supportreplication of human Ads. It is conceivable that chickens will rapidlyeliminate Ad5 after the immune repertoire is mobilized toward abeneficial immune protection following vaccination. ADNA-primed/Ad.5-boosted malaria vaccine successfully protected humansubjects against live malaria sporozoite challenge following mosquitofeeding. An Ad5-vectored, nasal vaccine was serendipitously shown toconfer rapid protection against influenza in a drug-like manner.Development of a drug-vaccine duo that consolidates drug and vaccineinto a single package that is not impaired by drug resistance wouldfundamentally change the way influenza drugs and vaccines are prepared.Overall, more and more adverse effects induced by systemic vaccinationhave been identified. Noninvasive mucosal immunization is safer and moreeffective in conferring protection against mucosal pathogens than itssystemic counterpart.

Tables

TABLE 2 Examples of protective immunity induced by adenovirus-vectoredvaccines against live pathogens in animal models Pathogen antigenexpressed from Vaccine Ad Route Animal model Challenge Ref. Ad26-Ebolavirus GP im. Nonhuman Ebolavirus [107] primed/Ad35- primate boostedAd5 Ebolavirus GP im. Nonhuman Ebolavirus [108] primate Ad5 AngolaMarburg im. Nonhuman Angola Marburg [109] virus GP primate virus BCG-Ag85A in., im. Guinea pig Mycobacterium [110] primed/Ad5- tuberculosisboosted Ad5 PA im. Mouse Bacillus anthracis [111] Sterne spore Ad5 Avianinfluenza in., im. Mouse Avian influenza [77, 78] virus H5 HA virusInfluenza vaccine- Influenza virus im. Mouse Heterosubtypic [112]primed/Ad5- HA influenza virus boosted Ad5 Avian influenza im., in ovo,Chicken Avian influenza [78, 97, virus H5 HA ovular virus 98, 102] Ad5Influenza virus im. Pig Swine influenza [104] HA/nucleoprotein virus Ad5Hantavirus im. Hamster Hantavirus [113] nucleocapsid/GP Ad5 Botulinumin, Mouse Botulinum [114] neurotoxin neurotoxin C-fragment Ad5 Measlesvirus in, im. Cotton rat Measles virus [70] fusion protein/HA Canine AdRabies virus GP Oral Raccoon, skunk Rabies virus [106] Ad5 V antigen im.Mouse Yersinia pestis [115] Ad: Adenovirus; BCG: BacillusCalmette-Guérin; GP: Glycoprotein; HA: Hemagglutinin; im.:Intramuscular; in.: Intranasal; PA: Protective antigen.

TABLE 3 Examples of human clinical trials of adenovirus andadenovirus-vectored vaccines Pathogen antigen Vaccine expressed from AdRoute Challenge Ref. Ad4 and Ad7 None Oral Natural infection by [31] AdDNA-primed/Ad5- CSP/AMA1 im. Malaria sporozoite [64] boosted Ad5 HIV-1gag/pol/nef im. Natural infection by [58, 59] HIV-1 DNA-primed/Ad5-HIV-1 gag/pol/env im. None [203] boosted Ad5-primed/Ad5- HIV-1 gag im.None [203] boosted Ad5-primed/NYVAC- HIV-1 gag/pol/env/nef im. None[203] boosted; NYVAC- primed/Ad5-boosted Ad5 Influenza virus H1 in. andNone [203] HA skin patch Ad4-primed/Ad4- Avian influenza virus Oral None[203] boosted H5 HA Encapsulated and Avian influenza virus Oral None[203] adjuvanted Ad5 H5 HA Ad5 Mycobacterium im. None [203] tuberculosis85A BCG-primed/Ad35- M. tuberculosis im. None [203] boosted 85A/85B/10.4Ad: Adenovirus; AMA1: Apical membrane antigen 1; BCG: BacillusCalmette-Guérin; CSP: Circumsporozoite protein; HA: Hemagglutinin; im.:Intramuscular; in.: Intranasal.

TABLE 4 Rational to develop an adenovirus serotype 5 vectored influenzadrug-vaccine duo in light of licensed influenza vaccines. Requirement touse Concomitant Requirement embryonated use with to propagate chickeneggs Replication licensed an influenza as the Production post- Mode ofinfluenza Vaccine virus substrate speed administration administrationdrugs TIV Yes Yes Slow No** Needle injection Yes** LAIV* Yes Yes SlowYes Nasal Spray** No DVD [89] No** No** Fast** No** Nasal Spray** Yes**Systemic Adverse effects inflammation; Nearly Broad following co-Reassortment platelet reactivity; immediate protection administrationwith a wild cardia autonomic protection against with undetectableinfluenza dysfunction; against heterosubtypic contaminating virusnarcolepsy influenza strains pathogens No** Yes [2, 201] No No Can beserious Yes No** No No Mild to none** No** No** Yes** Yes** Mild tonone** When an adenovirus serotype 5 (Ad5)-vectored influenza DVD isdeveloped, the multiple problems associated with propagating a liveinfluenza virus in qualified chicken eggs [35] can be eliminated.**Desirable outcomes. *Known as FluMist ® in the USA; a LAIV produced inMadin Darby canine kidney (MDCK) cells was licensed in Europe althoughits licensure in the USA has not been approved by the US FDA due to MDCKcalls' association with tumorigenicity and oncogenicity [116]. DVD:Drug-vaccine due; LAIV: Live-attenuated influenza virus vaccine; TIV:Trivalent inactivated influenza virus vaccine.

TABLE 5 Rationale to develop an Ad-5 vectored influenza drug-vaccine duein light of licensed influenza drugs. M2 ion Drug-vaccine channelNeuraminidase duo Associated problems blocker inhibitor [89] Requirementto administer Yes Yes No** multiple doses Potential to induce drug Yes[95] Yes [95] No** resistance Prophylactic therapy Yes** Yes** Yes**Postexposure therapy Partial** Partial** No Regulation of mucosalUnknown Oseltamivir Enhances immunity suppresses mucosal mucosalprotective immunity in immunity animals [96] [89]** Sustained protectionNo No Yes** over a few months **Desirable outcomesThe principle reason to develop an Ad5-vectored influenza drug-vaccineduo (DVD) in light of licensed infleunza drugs is its potential not tobe impaired by drug resistance, in addition to the convenience toconsolidate drug and vaccine into a single package [89]. Please notethat the development of DVD is still in its early stages; whetherprophylactic therapy can be reproduced in human subjects and whetherpost-exposure therapy can be developed remain to be seen. M2 ion channelblockers includes amantadine and rimantadine; neuraminidase inhibitorincludes oseltamivir (Tamiflu) and zanamivir (Relenza); DVD representsAd5-vectored influenza drug-vaccine duo [89].

Information Resources

-   Van Kampen K R, Shi Z, Gao P et al. Safety and immunogenicity of    adenovirus-vectored nasal and epicutaneous influenza vaccines in    humans. Vaccine 23, 1029-1036 (2005).-   Toro H, Tang D C, Suarez D L, Sylte M J, Pfeiffer J, Van Kampen K R.    Protective avian influenza in ovo vaccination with nonreplicating    human adenovirus vector. Vaccine 25, 2886-2891 (2007).-   Zhang J, Tarbet E B, Feng T, Shi Z, Van Kampen K R, Tang D C.    Adenovirus-vectored drug-vaccine duo as a rapid-response tool for    conferring seamless protection against influenza. PLoS ONE 6, e22605    (2011).

REFERENCES

-   1. Salomon M E, Halperin R, Yee J. Evaluation of the two-needle    strategy for reducing reactions to DPT vaccination. Am. J. Dis.    Child. 141, 796-798 (1987).-   2. Lanza G A, Barone L, Scalone G et al. Inflammation-related    effects of adjuvant influenza A vaccination on platelet activation    and cardiac autonomic function. J. Intern. Med. 269, 118-125 (2011).-   3. Jae S Y, Heffernan K S, Park S H et al. Does an acute    inflammatory response temporarily attenuate parasympathetic    reactivation? Clin. Auton. Res. 20, 229-233 (2010).-   4. Sever J L, Brenner A I, Gale A D et al. Safety of anthrax    vaccine: an expanded review and evaluation of adverse events    reported to the Vaccine Adverse Event Reporting System (VAERS).    Pharmacoepidemiol. Drug Saf. 13, 825-840 (2004).-   5. Minor P. Vaccine-derived poliovirus (VDPV): impact on    poliomyelitis eradication Vaccine 27, 2649-2652 (2009).-   6. Poland G A, Grabenstein J D, Neff J M. The US smallpox    vaccination program: a review of a large modern era smallpox    vaccination implementation program. Vaccine 23, 2078-2081 (2005).-   7. Lewis D J, Huo Z, Barnett S et al. Transient facial nerve    paralysis (Bell's palsy) following intranasal delivery of a    genetically detoxified mutant of Escherichia coli heat labile toxin.    PLoS ONE 4, e6999 (2009).-   8. Couch R B. Nasal vaccination, Escherichia coli enterotoxin, and    Bell's palsy. N. Engl. J. Med. 350, 860-861 (2004).-   9. Carlson B C, Jansson A M, Larsson A, Bucht A, Lorentzen J C. The    endogenous adjuvant squalene can induce a chronic T-cell-mediated    arthritis in rats. Am. J. Pathol. 156, 2057-2065 (2000).-   10. Finch C E, Crimmins E M. Inflammatory exposure and historical    changes in human life-spans. Science 305, 1736-1739 (2004).-   11. Gregor M F, Hotamisligil G S. Inflammatory mechanisms in    obesity. Annu. Rev. Immunol, 29, 415-445 (2011).-   12. O'Callaghan D S, O'Donnell D, O'Connell F, O'Byrne K J. The role    of inflammation in the pathogenesis of non-small cell lung    cancer. J. Thorac. Oncol. 5, 2024-2036 (2010).-   13. Witte M F, Geurts J J, de Vries E1 E, van der Valk P, van    Horssen J. Mitochondrial dysfunction: a potential link between    neuroinflammation and neurodegeneration? Mitochondrion 10, 411-418    (2010).-   14. Gallichan W S, Rosenthal K L. Long-lived cytotoxic T lymphocyte    memory in mucosal tissues after mucosal but not systemic    immunization. J. Exp. Med. 184, 1879-1890 (1996).-   15. Saurer L, McCullough K C, Summerfield A. In vitro induction of    mucosa-type dendritic cells by all-trans retinoic acid. J. Immunol.    179, 3504-3514 (2007).-   16. Molenaar R, Greuter M, van der Marel A P et al. Lymph node    stromal cells support dendritic cell-induced gut-homing of T    cells. J. Immunol. 183, 6395-6402 (2009).-   17. Tang D C, Van Kampen K R. Toward. the development of vectored    vaccines in compliance with evolutionary medicine. Expert Rev.    Vaccines 7(4), 399-402 (2008).-   18. Karron R A, Talaat K, Luke C et al. Evaluation of two live    attenuated cold adapted H5N1 influenza virus vaccines in healthy    adults. Vaccine 27, 4953-4960 (2009).-   19. Van Kampen K R, Shi Z, Gao P et al. Safety and irnmunogenicity    of adenovirus vectored nasal and epicutaneous influenza vaccines in    humans. Vaccine 23, 1029-1036 (2005).-   20. Tang D C, Shi Z, Curiel D T. Vaccination onto bare skin. Nature    388, 729-730 (1997).-   21. Zhang J, Shi Z, Kong F K et al. Topical application of    Escherichia coli-vectored vaccine as a simple method for eliciting    protective immunity. Infect. Immun. 74, 3607-3617 (2006).-   22. Glenn G M, Taylor D N, Li X et al. Transcutaneous immunization:    a human vaccine delivery strategy using a patch. Nat. Med. 6,    1403-1406 (2000).-   23. Sullivan S P, Koutsonanos D G, del Pilar Martin M et al.    Dissolving polymer microneedle patches for influenza vaccination.    Nat. Med. 16, 915-920 (2010).-   24. Kaplan D H. In vivo function of Langerhans cells and dermal    dendritic cells. Trends Immunol. 31, 446-451 (2010).-   25. Soloff A C, Barratt-Bayes S M. !Enemy at the gates: dendritic    cells and immunity to mucosal pathogens. Cell Res. 20, 872-885    (2010).-   26. Brandes M, Willimann K, Moser B. Professional    antigen-presentation function by human gammadelta T cells. Science    309, 264-268 (2005). 27. Feng T, Cong Y, Qin H, Benveniste E N,    Elson C O. Generation of mucosal dendritic cells from bone marrow    reveals a critical role of retinoic acid. J. Immunol. 185, 5915-5925    (2010).-   28. Kaufman D R, Bivas-Benita M, Simmons N L, Miller D, Barouch D H.    Route of adenovirus-based HIV-1 vaccine delivery impacts the    phenotype and trafficking of vaccine-elicited CD8+ T lymphocytes. J.    Virol. 84, 5986-5996 (2010)-   29. San Martin C, Burnett R M. Structural studies on adenoviruses.    Curr. Top. Microbiol. Immunol. 272, 57-94 (2003).-   30. Lichtenstein D L, Wold W S. Experimental infections of humans    with wild-type adenoviruses and with replication competent    adenovirus vectors: replication, safety, and transmission. Cancer    Gene Ther. 11, 819-829 (2004).-   31. Howell M R, Nang R N, Gaydos C A, Gaydos J C. Prevention of    adenoviral acute respiratory disease in Army recruits:    cost-effectiveness of a military vaccination policy. Am. J. Prey.    Med. 14, 168-175 (1998).-   32. Haj-Ahmad Y, Graham F L. Development of a helper-independent    human adenovirus vector and its use in the transfer of the herpes    simplex virus thymidine kinase gene. J. Virol. 57, 267-274 (1986).-   33. Zhu J, Grace M, Casale J et al. Characterization of    replication-competent adenovirus isolates from large-scale    production of a recombinant adenoviral vector. Hum. Gene Ther. 10,    113-121 (1999).-   34. Fallaux F J, Bout A, van der Vlede I et al. New helper cells and    matched early region 1-deleted adenovirus vectors prevent generation    of replication-competent adenoviruses. Hum. Gene Ther. 9, 1909-1917    (1998).-   35. Tang D C, Zhang J, Toro H, Shi Z, Van Kampen K R. Adenovirus as    a carrier for the development of influenza virus-free avian    influenza vaccines. Expert Rev. Vaccines 8, 469-481 (2009).-   36. Wang L, Cheng C, Ko S Y et al. Delivery of human    immunodeficiency virus vaccine vectors to the intestine induces    enhanced mucosal cellular immunity. J. Virol. 83, 7166-7175 (2009).-   37. Tang J, Olive M, Champagne K et al. Adenovirus hexon epitope is    recognized by most adults and is restricted by HLA DP4, the most    common class II allele, Gene Ther. 11, 1408-1415 (2004).-   38. Nwanegbo E, Vardas E, Gao W et al. Prevalence of neutralizing    antibodies to adenoviral serotypes 5 and 35 in the adult populations    of The Gambia, South Africa, and the United States. Clin. Diagn,    Lab. Immunol, 11, 351-357 (2004).-   39. Barouch D H, Kik S V, Weverling G J et al. International    seroepidemiology of adenovirus serotypes 5, 26, 35, and 48 in    pediatric and adult populations. Vaccine 29, 5203-5209 (2011).-   40. Yang Y, Nunes F A, Berencsi K et al. Cellular immunity to viral    antigens limits E1-deleted adenoviruses for gene therapy. Proc. Natl    Acad, Sci, USA 91, 4407-4411 (1994).-   41. Croyle M A, Patel A, Tran K N et al. Nasal delivery of an    adenovirus-based vaccine bypasses pre-existing immunity to the    vaccine carrier and improves the immune response in mice. PLoS ONE    3, e3548 (2008).-   42. Stone D, Liu Y, Li Z Y et al. Comparison of adenoviruses from    species B, C, E, and F after intravenous delivery. Mol. Ther. 15,    2146-2153 (2007).-   43. Sharma A, Bangari D S, Tandon M et al. Comparative analysis of    vector biodistribution, persistence and gene expression following    intravenous delivery of bovine, porcine and human adenoviral vectors    in a mouse model. Virology 386, 44-54 (2009).-   44. Roy S, Medina-Jaszek A, Wilson M J et al. Creation of a panel of    vectors based on ape adenovirus isolates. J. Gene Med. 13, 17-25    (2011). 45. Patel A, Tikoo S, Kobinger G. A porcine adenovirus with    low human seroprevalence is a promising alternative vaccine vector    to human adenovirus 5 in an H5N1 virus disease model. PLoS ONE 5,    e15301 (2010).-   46. Chen E C, Yagi S, Kelly K R et al. Crossspecies transmission of    a novel adenovirus associated with a fulminant pneumonia outbreak in    a new world monkey colony. PLoS Pathog. 7, e1002155 (2011).-   47. Seto J, Walsh M P, Metzgar D, Seto D. Computational analysis of    adenovirus serotype 5 (HAdV-C5) from an HAdV coinfection shows    genome stability after 45 years of circulation. Virology 404, 180186    (2010).-   48. Boyer J L, Sofer-Podesta C, Ang J et al. Protective immunity    against a lethal respiratory Yersinia pestis challenge induced by V    antigen or the F1 capsular antigen incorporated into adenovirus    capsid. Hum. Gene Ther. 21, 891-901 (2010).-   49. Worgall S, Krause A, Qiu J et al. Protective immunity to    Pseudomonas aeruginosa induced with a capsid-modified adenovirus    expressing P. aeruginosa OprF. J. Virol. 81, 13801-13808 (2007).-   50. Osada T, Yang X Y, Hartman Z C et al. Optimization of vaccine    responses with an E1, E2b and E3-deleted Ads vector circumvents    pre-existing anti-vector immunity. Cancer Gene Ther. 16, 673-682    (2009).-   51. Gabitzsch E S, Xu Y, Yoshida L H et al. Novel adenovirus type 5    vaccine platform induces cellular immunity against HIV-1 Gag, Pol,    Nef despite the presence of Ads immunity, Vaccine 27, 6394-6398    (2009).-   52. Parks R J, Chen L, Anton iv1 et al, A helper-dependent    adenovirus vector system: removal of helper virus by Cre-mediated    excision of the viral packaging signal. Proc. Nail Acad. Sci, USA    93, 13565-13570 (1996).-   53. Shi Z, Zeng M, Yang G et al. Protection against tetanus by    needle-free inoculation of adenovirus-vectored nasal and    epicutaneous vaccines. J. Virol. 75, 11474-11482 (2001).-   54. Song K, Bolton D L, Wilson R L et al. Genetic immunization in    the lung induces potent local and systemic immune responses. Proc.    Nati Acad. Sci. USA 107, 22213-22218 (2010).-   55. Raper S E, Chirmule N, Lee F S et al, Fatal systemic    inflammatory response syndrome in a ornithine transcarbamylase    deficient patient following adenoviral gene transfer. Mol. Genet.    Metab. 80, 148-158 (2003).-   56. Hartman Z C, Appledore D M, Amalfitano A. Adenovirus vector    induced innate immune responses: impact upon efficacy and toxicity    in gene therapy and vaccine applications. Virus Res. 132, 1-14    (2008).-   57. Higashimoto Y, Yamagata Y, Itoh H. Complex effect of adenovirus    early region proteins on innate immune system. Inflamm. Allergy Drug    Targets 5, 229-237 (2006).-   58. Buchbinder S P, Mehrotra D V, Duerr A et al. Efficacy assessment    of a cell-mediated immunity HIV-1 vaccine (the Step study): a    double-blind, randomised, placebo controlled, test-of-concept trial.    Lancet 372, 1881-1893 (2008).-   59. McElrath M J, De Rosa S C, Moodie Z et al. HIV-1 vaccine-induced    immunity in the test-of-concept Step Study: a case-cohort analysis.    Lancet 372, 1894-1905 (2008).-   60. Appledorn D M, Aldhamen Y A, Godbehere S, Seregin S S,    Amalfitano A, Sublingual administration of an adenovirus serotype 5    (Ad5)-based vaccine confirms Toll-like receptor agonist activity in    the oral cavity and elicits improved mucosal and systemic    cell-mediated responses against HIV antigens despite preexisting Ad5    immunity. Clin. Vaccine Immunol. 18, 150-160 (2011).-   61. Benlahrech A, Harris J, Meiser A et al. Adenovirus vector    vaccination induces expansion of memory CD4 T cells with a mucosal    homing phenotype that are readily susceptible to HIV-1. Proc. Natl    Acad. Sci. USA 106, 19940-19945 (2009).-   62. Joseph A, Itskovitz-Cooper N, Samira S et al. A new intranasal    influenza vaccine based on a novel poly cationic lipid-ceramide    carbamoyl-spermine (CCS) I. Immunogenicity and efficacy studies in    mice. Vaccine 24, 3990-4006 (2006).-   63. Wang J, Thorson L, Stokes R W et al. Single mucosal, but not    parenteral, immunization with recombinant adenoviral based vaccine    provides potent protection from pulmonary tuberculosis. J. Immunol.    173, 6357-6365 (2004).-   64. Ockenhouse C. Prime boost regimens of DNA and    adenovirus-vectored malaria vaccines: lessons learned from    preclinical and clinical studies. Presented at: Conference of Gene    Based Vaccines. Vienna, Austria, 13-14 Sep. 2010,-   65. McWilliam A S, Nelson D J, Holt P G. The biology of airway    dendritic cells. Immunol. Cell Biol. 73, 405-413 (1995).-   66. Brandtzaeg P. Potential of nasopharynx associated lymphoid    tissue for vaccine responses in the airways. Am. J. Respir, Crit.    Care Med. 183, 1595-1604 (2011).-   67. Lubeck M D, Natuk R J, Chengalvala M et al. Immunogenicity of    recombinant adenovirus-human immunodeficiency virus vaccines in    chimpanzees following intranasal administration. AIDS Res. Hum.    Retroviruses 10, 1443-1449 (1994).-   68. Lemiale F, Kong W P, Akyurek L M et al. Enhanced mucosal    immunoglobulin A response of intranasal adenoviral vector human    immunodeficiency virus vaccine and localization in the central    nervous system. J. Virol. 77, 10078-10087 (2003).-   69. Gallichan W S, Rosenthal K L. Long-term immunity and protection    against herpes simplex virus type 2 in the murine female genital    tract after mucosal but not systemic immunization. J. Infect. Dis.    177, 1155-1161 (1998).-   70. Lobanova L M, Baig T T, Tikoo S K, Zakhartchouk A N. Mucosal    adenovirus-vectored vaccine for measles. Vaccine 28, 7613-7619    (2010).-   71. del Rio M L, Bernhardt G, Rodriguez-Barbosa J I, Forster R.    Development and functional specialization of CD103+ dendritic cells.    Immunol. Rev. 234, 268-281 (2010).-   72. Zhang Z, Krimmel J, Zhang Z, Hu Z, Seth P. Systemic delivery of    a novel liverdetargeted oncolytic adenovirus causes reduced liver    toxicity but maintains the antitumor response in a breast cancer    bone metastasis model. Hum. Gene Ther. (In Press) (2011).-   73. Li C, Ziegler R J, Cherry M et al. Adenovirus-transduced lung as    a portal for delivering alpha-galactosidase A into systemic    circulation for Fabry disease. Mol. Ther. 5, 745-754 (2002).-   74. Toovey S. Influenza-associated central nervous system    dysfunction: a literature review. Travel Med. Infect. Dis. 6,    114-124 (2008).-   75. Carter N J, Curran M P. Live attenuated influenza vaccine    (FluMist®; Fluenz™): a review of its use in the prevention of    seasonal influenza in children and adults. Drugs 71, 1591-1622    (2011).-   76. Zhang S Y, Jouanguy E, Ugolini S et al. TLR3 deficiency in    patients with herpes simplex encephalitis. Science 317, 1522-1527    (2007).-   77. Hoelscher M A, Garg S, Bangari D S et al, Development of    adenoviral-vector-based pandemic influenza vaccine against    antigenically distinct human H5N1 strains in mice. Lancet 367,    475-481 (2006).-   78. Gao W, Soloff A C, Lu X et al. Protection of mice and poultry    from lethal H5N1 avian influenza virus through adenovirus-based    immunization. J. Virol. 80, 1959-1964 (2006).-   79. Xiang Z Q, Yang Y, Wilson J M, Ertl H C. A replication-defective    human adenovirus recombinant serves as a highly efficacious vaccine    carrier. Virology 219, 220-227 (1996).-   80. Tang D C, DeVit M, Johnston S A. Genetic immunization is a    simple method for eliciting an immune response. Nature 356,152-154    (1992).-   81. Jones S, Evans K, McElwaine-Johnn H et al. DNA vaccination    protects against an influenza challenge in a double-blind randomised    placebo-controlled Phase lb clinical trial. Vaccine 27, 2506-2512    (2009).-   82. Ulmer J B, Donnelly J J, Parker S E et al. Heterologous    protection against influenza by injection of DNA encoding a viral    protein. Science 259, 1745-1748 (1993).-   83. van Drunen Littel-van den Hurk S, Hannaman D. Electroporation    for DNA immunization: clinical application. Expert Rev. Vaccines    9(5), 503-517 (2010).-   84. Kutzler M A, Weiner D B. DNA vaccines: ready for prime time?    Nat. Rev. Genet. 9, 776-788 (2008).-   85. Peng Z. Current status of gendicine in China: recombinant human    Ad-p53 agent for treatment of cancers. Hum. Gene Ther, 16, 1016-1027    (2005).-   86. Evans R K, Nawrocki D K, Isopi L A et al. Development of suable    liquid formulations for adenovirus-based vaccines. J. Pharm. Sci.    93, 2458-2475 (2004).-   87. Alcock R, Cottingham M G, Bonier C S et al. Long-term    thermostabilization of live pox viral and adenoviral vaccine vectors    at supraphysiological temperatures in carbohydrate glass. Sci,    Transl. Med. 2, 19ra12 (2010).-   88. Croyle M A, Cheng X, Wilson J M. Development of formulations    that enhance physical stability of viral vectors for gene therapy.    Gene Ther. 8, 1281-1290 (2001).-   89. Zhang J, Tarbet E B, Feng T et al. Adenovirus-vectored    drug-vaccine duo as a rapid-response tool for conferring seamless    protection against influenza. PLoS ONE 6, e22605 (2011).-   90. Yamaguchi T, Kawabata K, Kouyama E et al. Induction of type I    interferon by adenovirus-encoded small RNAs. Proc. Natl Acad. Sci.    USA 107,17286-17291 (2010).-   91. Thiele A T, Sumpter T L, Walker J A et al. Pulmonary immunity to    viral infection: adenovirus infection of lung dendritic cells    renders T cells nonresponsive to interleukin-2. J. Virol. 80,    1826-1836 (2006).-   92. Zhu J, Huang X, Yang Y. A critical role for type I IFN-dependent    NK cell activation in innate immune elimination of adenoviral    vectors in vivo. Mol. Ther. 16, 1300-1307 (2008).-   93. Tuvim M J, Evans S E, Clement C G, Dickey B F, Gilbert B E.    Augmented lung inflammation protects against influenza A pneumonia.    PLoS ONE 4, e4176 (2009).-   94. Norton E B, Clements J D, Voss T G, Cardenas-Freytag L.    Prophylactic administration of bacterially derived immunomodulators    improves the outcome of influenza virus infection in a murine    model. J. Virol. 84, 2983-2995 (2010).-   95. Poland G A, Jacobson R M. Ovsyannikova I G. Influenza virus    resistance to antiviral agents: a plea for rational use. Clin.    Infect. Dis. 48, 1254-1256 (2009).-   96. Takahashi E, Kataoka K, Fujii K et al. Attenuation of inducible    respiratory immune responses by oseltamivir treatment in mice    infected with influenza A virus. Microbes Infect. 12, 778-783    (2010).-   97. Toro H, Tang D C, Suarez D L et al. Protective avian influenza    in ovo vaccination with non-replicating human adenovirus vector.    Vaccine 25, 2886-2891 (2007).-   98. Toro H, Tang D C, Suarez D L, Zhang J, Shi Z. Protection of    chickens against avian influenza with non-replicating    adenovirus-vectored vaccine. Vaccine 26, 2640-2646 (2008).-   99. Toro H, Tang D C. Protection of chickens against avian influenza    with non-replicating adenovirus-vectored vaccine. Poult. Sci. 88,    867-871 (2009).-   100. Avakian A P, Poston R M, Kong F K, Van Kampen K R, Tang D C.    Automated mass immunization of poultry: the prospect for    nonreplicating human adenovirus-vectored in ovo vaccines. Expert    Rev. Vaccines 6(3), 457-465 (2007).-   101. Singh S, Toro H, Tang D C et al. Nonreplicating adenovirus    vectors expressing avian influenza virus hemagglutinin and    nucleocapsid proteins induce chicken specific effector, memory and    effector memory CD8+ T lymphocytes. Virology 405, 62-69 (2010).-   102. Toro H, Suarez D L. Tang D C, van Ginkel F W, Breedlovea C.    Avian influenza mucosal vaccination in chickens with    replication-defective recombinant adenovirus vaccine. Avian Dis. 55,    43−47 (2011).-   103. van Ginkel F, Tang D C, Gulley S L, Toro H. Induction of    mucosal immunity in the avian Harderian gland with a replication    deficient Ad5 vector expressing avian influenza. H5 hemagglutinin.    Dev. Comp. Immunol. 33, 28-34 (2009).-   104. Wesley R D, Tang M, Lager K M. Protection of weaned pigs by    vaccination with human adenovirus 5 recombinant viruses expressing    the hemagglutinin and the nucleoprotein of H3N2 swine influenza    virus. Vaccine 22, 3427-3434 (2004).-   105. Toro H, van Ginkel W, Tang D C et al. Avian influenza    vaccination in chickens and pigs with replication-competent    adenovirus-free human recombinant adenovirus 5. Avian Dis. 54(1    Suppl.), 224-231 (2010).-   106. Henderson H, Jackson F, Bean K et al. Oral immunization of    raccoons and skunks with a canine adenovirus recombinant rabies    vaccine. Vaccine 27, 7194-7197 (2009).-   107. Geisbert T W, Bailey M, Hensley L et al. Recombinant adenovirus    serotype 26 (Ad26) and Ad35 vaccine vectors bypass immunity to Ad5    and protect nonhuman primates against ebolavirus challenge. J.    Virol. 85, 4222-4233 (2011).-   108. Pratt W D, Wang D, Nichols D K et al. Protection of nonhuman    primates against two species of Ebola virus infection with a single    complex adenovirus vector, Clin. Vaccine Immunol. 17, 572-581    (2010).-   109. Geisbert T W, Bailey M, Geisbert J B et al. Vector choice    determines irnmunogenicity and potency of genetic vaccines against    Angola Marburg virus in nonhuman primates. J. Virol. 84, 10386-10394    (2010).-   110. Xing Z, McFarland C T, Sallenave J M et al. Intranasal mucosal    boosting with an adenovirus-vectored vaccine markedly enhances the    protection of BCG-primed guinea pigs against pulmonary tuberculosis,    PLoS ONE 4, e5856 (2009).-   111. McConnell M J, Hanna P C, Imperiale M J. Adenovirus-based    prime-boost immunization for rapid vaccination against anthrax. Mol.    Ther. 15, 203-210 (2007).-   112. Wei C J, Boyington J C, McTamney P M et al. Induction of    broadly neutralizing H1N1 influenza antibodies by vaccination.    Science 329, 1060-1064 (2010).-   113. Safronetz D, Hegde N R, Ebihara H et al. Adenovirus vectors    expressing hantavirus proteins protect hamsters against lethal    challenge with andes virus. J. Virol, 83, 7285-7295 (2009).-   114. Xu Q, Pichichero M E, Simpson L L et al. An adenoviral    vector-based mucosal vaccine is effective in protection against    botulism. Gene Ther. 16, 367-375 (2009).-   115. Chiuchiolo M J, Boyer J L, Krause A et al. Protective immunity    against respiratory tract challenge with Yersinia pestis in mice    immunized with an adenovirus-based vaccine vector expressing V    antigen. J. Infect. Dis. 194, 1249-1257 (2006).-   116. Fox J L. FDA, producers moving toward mammalian cell-based flu    vaccines. Microbe 1, 54-55 (2006).-   117. Boyer J L, Kobinger G, Wilson J M, Crystal R G.    Adenovirus-based genetic vaccines for biodefense. Hum. Gene Ther.    16, 157-168 (2005).

Example 4 Adenovirus-Vectored InfluenzaRapid-and-Prolonged-Immunologicals-Therapeuticals

The goal of Ad5-vectored influenza RAPIT is to develop an influenzarapid-and-prolonged-immunologic-therapeutic (RAPIT) that can bemass-produced at low costs and mass-administered by non-medicalpersonnel; with the capability to confer rapid/sustained protectionagainst influenza but without the potential to induce drug resistanceand reassortment with a wild influenza virus. There is no requirement topropagate an influenza virus and no requirement for needle injection bylicensed medical personnel.

It is possible to rapidly generate Ad5-vectored influenza vaccinewithout growing influenza virus. In an influenza virus, growth variesfrom strain to strain, some strains are lethal, it is prone toreassortment and mutation events and there is low-titer protection ineggs. In an Ad vector encoding influenza HA, there are more consistentgrowth rates, the vector is benign, there are no reassortment events,there is high-titer production in PER.C6 cells and a new RCA-free Ad canbe generated by the AdHigh system within one month.

Ad5-vectored influenza vaccines in cultured suspension cells may be massproduced. For an Ad-vectored flu vaccine, cloning of influenza HA intoAd does not require growth of influenza virus, a 500-liter wavebioreactor can produce 10¹⁶ Ad particles at one time from PER.C6suspension cells in serum-free medium, Ad particles can be purified bycolumn chromatography and production of Ad-vectored flu vaccines can bestreamlined in rapid response to an escalation in demand (FIG. 11). Fora conventional flu vaccine, some influenza virus strains do not growwell in eggs, the average yield is approximately one dose per egg,contamination is more difficult to identify in eggs than in cellcultures, there may be egg-associated allergies and the processing iscumbersome.

Ad5-mediated gene therapy and nasal vaccination may be compared asfollows. In gene therapy, a therapeutic protein is expressed from Ad anda biological effect is induced directly by a correct dose of therapeuticprotein expressed from Ad in transduced cells. In nasal vaccination, theantigen protein is expressed from Ad, the antigen is presented and animmune response is induced through a cascade of reactions triggered byantigen expressed from Ad in transduced cells. Reports in support of thehypothesis that preexisting immunity to Ad does not interfere with thepotency of Ad-vectored nasal vaccines include Shi Z et al. J. Virol. 75:11474, 2001 (mice), Hoelscher M A et al. Lancet 367: 475, 2006 (mice),Croyle M A et al. PLoS ONE 3: e3548, 2008 (mice), Song K et al. PNAS107: 22213, 2010 (macaques) and Van Kampen K R et al. Vaccine 23: 1029,2005 (humans),

The study design of a human phase I clinical trial of an Ad5-vectorednasal avian influenza vaccine was as follows. An AdhVN1203/04.H5 vectorencoded HA1+HA2 of the A/VN/1203/04 (H5N1) avian influenza virus. Thestudy was a randomized, double-blind, placebo-controlled, single-sitestudy. There were three cohorts at an escalating dose of 10⁸, 10⁹, and10¹⁰ vp. The doses were administered by nasal spray and two doses onDays 0 and 28. There was a total of 48 healthy volunteers, aged 19-49.There were sixteen human subjects per dose cohort, including 4 placebocontrols per cohort. The cell culture was a RCA free, cell culture basedmanufacturing in PER.C6 suspension cells in serum-free medium. Theadverse events in the respiratory system in 30% or more of subjectsincluded rhinorrhea, nasal irritation, nasal congestion, cough and/orsore throat.

Example 5 Adenovirus Particle as a Broad-SpectrumRapid-and-Prolonged-Immunologic-Therapeutic (RAPIT) Against RespiratoryPathogens

FIG. 15 depicts prophylactic anthrax therapy by intranasal instillationof adenovirus particles shortly before spore challenge.

Methods. AdE (E1/E3-defective Ad5 empty vector without transgene) andAdVAV (E1/E3-defective Ad5 vector encoding Bacillus anthracis protectiveantigen) particles were intranasally (i.n.) administered dropwise intothe nostrils of young (2-month-old) female A/J mice in a volume of 0.05ml in a single-dose regimen shortly before i.n. challenge with 1×10⁵ cfu(˜25×LD₅₀) of Bacillus anthracis Sterne spores. Challenged animals weremonitored for survival on a daily basis for 14 days.

Results. AdVAV particles administered 2 days prior to challengeprotected 67% of mice against anthrax; AdE particles administered 2 daysprior to challenge protected 30% of mice against anthrax; AdE particlesadministered 1 day prior to challenge protected 22% of mice againstanthrax; untreated control mice and mice administered with diluted. AdEparticles all succumbed to anthrax within 5 days. AdVAV/−2, AdVAVparticles i.n. instilled 2 days prior to challenge at a dose of 1.3×10⁸ifu; AdE/−2, AdE particles i.n. instilled 2 days prior to challenge at adose of 1.3×10⁸ ifu; AdE*/−2, AdE particles i.n. instilled 2 days priorto challenge at a dose of 1.3×10⁶ ifu (100-fold dilution in PBS);AdE/−1, AdE particles i.n. instilled 1 day prior to challenge at a doseof 1.3×10⁸ ifu; Control, untreated control mice; numbers in parenthesesrepresent the number of animals in each group.

Significance. Data suggest that AdE or AdVAV particles may conferprophylactic anthrax therapy in a drug-like manner, probably byactivating a specific arm of innate immunity that impedes growth ofBacillus anthracis in infected animals. Data suggest that the PA geneexpressed from AdVAV may confer synergy with AdE-mediated protectionagainst anthrax. It is conceivable that nasal spray of AdVAV particlesmay confer more rapid protection against anthrax than other anthraxvaccines during a crisis.

FIG. 16 depicts post-exposure anthrax therapy by i.n. instillation ofAdVAV particles.

Methods. AdVAV particles were i.n. administered dropwise into thenostrils of young (2-month-old) female A/J mice in a volume of 0.05 mlin a single-dose regimen, either before or after i.n. challenge with4×10⁵ cfu (˜100×LD₅₀) of Bacillus anthracis Sterne spores. Ciprofloxacinwas administered by i.p. injection at a dose of 30 mg/kg (1 injectionper day for 2 days; injected 1 and 24 hours post-challenge). Challengedanimals were monitored for survival on a daily basis for 14 days.

Results. AdVAV particles administered 2 days prior to challengeprotected 40% of mice against anthrax nfirmation of FIGS. 2 and 15results); AdVAV particles administered 1 hour post-challenge delayeddeath but failed to improve survival rate; ciprofloxacin injected 1 hourpost-challenge also delayed death without success in improving survivalrate; AdVAV particles administered in conjunction with ciprofloxacininjection 1 hour post-challenge protected 56% of mice against anthrax;all untreated control mice died within 5 days. AdVAV/D-2, A.dVAVparticles i.n. instilled 2 days prior to challenge at a dose of 1.3×10⁸ifu; AdVAV/D0, AdVAV particles i.n. instilled 1 hour post-challenge at adose of 1.3×10⁸ ifu; AdVAV/Cipro/D0, AdVAV particles i.n. instilled 1hour post-challenge at a dose of 1.3×10⁸ ifu in conjunction with i.p.injection of ciprofloxacin; Cipro/D0, i.p. injection of ciprofloxacin;Control, untreated control mice without treatments prior to challenge;numbers in parentheses represent the number of animals in each group.

Significance. Data suggest that AdVAV particles may confer post-exposureanthrax therapy in conjunction with antibiotic treatments. Synergybetween AdVAV and antibiotics was revealed in this experiment. It isconceivable that nasal spray of AdVAV particles may be able to reducethe requirement for antibiotic use in a post-exposure setting.

Example 6 Adenovirus Particle as a Broad-SpectrumRapid-and-Prolonged-Immunologic-Therapeutic (RAPIT) Against RespiratoryPathogens

Recently, it has been demonstrated that intranasal (i.n.) administrationof ΔE1E3 adenovirus type 5 (Ads) particles, with or without a pathogenantigen encoded in the Ads genome, can confer prophylactic therapyagainst influenza before adaptive immunity is elicited. An Ads vectorencoding pathogen antigens may thus induce rapid and sustained seamlessprotection against a pathogen as a drug-vaccine duo (DVD). It has beendocumented that administration of ΔE1E3 Ads particles into mice rapidlyinduces the production of a wide array of inflammatory cytokines andchemokines including type I interferon (IFN-α and IFN-β); impairs lungdendritic cells; activates natural killer cells; induces production ofthe antiviral nitric oxide; triggers multi-faceted interactions betweenAd5 and blood proteins, platelets, macrophages, endothelial cells, andrespective parenchymal cells. It is conceivable that multiple reactionsinduced by the ΔE1E3 Ad5 particles may combine to establish ananti-influenza state in the airway, thus creating a multidimensionaldefense barrier that can hardly be bypassed by an influenza virus. It isunlikely that influenza is the only disease that can be arrested by Adsparticles; it is also unlikely that Ad5 particles can counteract alldiseases as a panacea. The findings merely show that a single i.n.administration of AdE particles can confer prophylactic therapy againstat least a subset of mucosal respiratory pathogens for many weeks inmice and use of the DVD should be unable to induce drug resistancebecause AdE particles change the habitat in the airway without directlyconferring mutational pressure to other viruses. Subsequent elicitationof sustained protective immunity by the DVD's vaccine componentfortifies efficacy. The development of a DVD platform will conceivablyfoster the development of novel clinical strategies in a wide variety ofdisease settings.

The goal of this Example is to evaluate prophylactic intranasaltreatment with Vaxin's AdE (Ad5 empty vector without an RSV transgene)on respiratory syncytial virus (RSV)-infected cotton rats (CR). Theendpoints of this study are the demonstration of reduced virus titers inthe lung lavage (3 mL) and nasal wash (2 mL) fluids of the infectedcotton rats (ca. 60-125 gm in weight) compared to untreated cotton rats.Virus quantification will be done by plaque reduction assay. FIG. 17depicts the effect of AdE administered intranasally on RSV-Tracy nasalwash as well as lung lavage virus titers on Day +4.

Prophylactic Effectiveness in the RSV-Cotton Rat Model: Cotton Rats(60-125 gm body weight):

Group 1: 6 CR prophylactically (day −2) treated intranasally withvehicle (A195 buffer).

Group 2: 6 CR prophylactically (day −30) treated intranasally with2.4×10⁸ ifu of AdE.

Group 3: 6 CR prophylactically (day −2) treated intranasally with2.4×10⁸ ifu of AdE.

Group 4: 6 CR prophylactically (days −30 and −2) treated intranasallywith 2.4×10⁸ ifu of AdE during each treatment cycle (prime/boost)

Group 5: 6 CR prophylactically (−5 h) treated intranasally with 2.4×10⁸ifu of AdE.

Challenge Virus: RSV-Tracy (P3 w.p. 1/20/12 grown in HEp-2 cells)2.25×10⁵ PFU intranasally (100 μL) to cotton rats (60-125 gm) lightlyanesthesized with isoflurane. Stock: 2.25×10⁶ PFU/mL.

AdE vector: Vehicle (A195 buffer) AdE at concentrations of 2.4×10⁹ arestored at −80° C. Just before use, materials are warmed to roomtemperature. At least 0.8 mL of each treatment for each group (6CR/group×0.1 mL of inoculum) is needed. Unused material is kept at −80°C.

Collection of organs and samples. Following euthanasia with CO₂, eachcotton rat are weighed and the sex and age recorded. The left and one ofthe large right lobes of the lungs will be removed, rinsed in sterilewater to remove external blood contamination and weighed. The left lobeis transpleurally lavaged using 3 mL of Iscove's media with 15% glycerinmixed with 2% FBS-MEM (1:1, v:v) in a 3 mL syringe with a 26 g ⅜ needleand injecting at multiple sites to totally inflate the lobe. Lavagefluid is recovered by gently pressing the inflated lobe flat and used totranspleurally lavage the right lobe following the same technique. Thelavage fluid is collected and stored on ice until titered. For nasalwashes of the upper respiratory tract, the jaws are disarticulated. Thehead is then be removed and 1 mL of Iscove's media with 15% glycerinmixed with 2% FBS-MEM (1:1, v:v) aare pushed through each nare (total of2 mL). The effluent is collected from the posterior opening of thepallet and stored on ice until titered. Samples are not frozen beforetitration which occurs at the end of sample collecting.

RSV Tracy lung lavage titers (PFU/gm lung) and nasal wash titers (totalPFU). Plaque assays are performed using 24-well tissue cultures platescontaining nearly confluent monolayers (20 to 40×10⁴ cells/well) ofHEp-2 cells prepared in 10% FCS 24 hr prior to start of assay. At thestart of each assay, dilutions (usually serial log 10) are made of thetest samples. A 0.2 mL sample of each is then be added to wells induplicate and allowed to adsorb for 90 min with occasional gentleagitation. After the inoculum is removed, the monolayers is thenoverlayed with 0.75% methylcellulose in 2% FBS-MEM containingantibiotics, vitamins and other nutrients. Tissue culture and positivevirus controls are included in each assay. The plates is placed in a 36°C., 5% CO₂ incubator. Day 6±1 day later, plates are stained with 0.1%crystal violet/10% formalin solution (1.5 mL/well) and allowed to sitfor 24-48 hr at room temperature. Wells are rinsed with water. Plaqueswhen present are easily visible (clear circles on a very dark bluebackground). All of the plaques in wells containing between 20 and 80plaques will be enumerated, averaged and the virus titers calculated astotal log 10 PFU for nasal wash fluid or log 10 PFU/g of tissue forlungs or other organs. The lower limit of detection by this method isapproximately 1.5 log 10 PFU/g tissue.

Antibody Response to AdE: Blood is collected from the orbital plexusfrom Groups 2 and 4 (3 CR/group) on day −30 and Groups 2 and 4 (6CR/group) on day −2. Blood will be collected from Groups 1-5 on Day +4.Serums are stored at −20° C.

Reserve samples. Aliquots of nasal wash and lung lavage fluids (Groups1-5) are saved, stored at −80° C. Serum samples from day +4 are saved,stored at −80° C.

TABLE 6 Proposed plan of study AdE Volume Particles Treatment Group¹Treatment² Route (mL) (ifu/CR) Schedule Harvest Endpoint 1 Buffer Day −2— 0 0 Day −2 Day +4 Virus titer in 2 AdE, Day −30 i.n. 0.100 2.4 × 10⁸Day −2 lung lavage 3 AdE, Day −2 i.n. 0.100 2.4 × 10⁸ Day −30 and nasalwash 4 AdE, Days −30, −2 i.n. 0.100 2.4 × 10⁸ Days −30 and −2 fluids byPFU 5 AdE, −5 Hours i.n. 0.100 2.4 × 10⁸ Hour −5 Abbreviations: i.n.,intranasal; PFU, plaque forming units, ¹N = 6 animals/group; 30 animalstotal. ²All animals to be challenged i.n. (100 μl) with RSV-Tracey (ca.2.25 × 10⁵ PFU) on day 0.

TABLE 7 Daily Schedule: Wednesday Wednesday Friday Saturday- Tuesday Day−30 Day −2 Day 0 Monday Day +4 Days Jan. 18, 2012 February 15 February17 Day +1 to +3 February 21 +5 to +16 Treat Groups 2 Treat Group 1 At −5h, treat Monitor Collect nasal Monitor and 4 with i.n. with i.n.Vehicle; Group 5 with i.n. animals wash and lavage titrations AdE;Groups 3 and 4 AdE 2 lobes of lungs for PFU Bleed Groups 2 with i.n.AdE; At 0 h, infect for virus titers; and 4 (3 CR/gp) Bleed Groups 2Groups 1-5 i.n. Collect blood and 4 (6 CR/gp) with RSV-Tracy from Groups1-5

Abbreviations: i.n., intranasal; PFU, plaque forming units; gp, group.

Timeline Day 0:

9 am=>Treat group 5 i.n. AdE

2 pm=>Infect all groups

Dosage and Lung and Body Weights on Day +4.

TABLE 8 Lung and Body Weights on Day +4 AdE Lung Lobe Body Weight DoseWeight (g)¹ (g)² Group Treatment (ifu/cr) Mean SD Mean SD 1 Buffer Day−2 0 0.31 0.02 163.0 16.1 2 AdE, Day −30 2.4 × 10⁸ 0.35 0.05 155.9 12.13 AdE, Day −2 2.4 × 10⁸ 0.36 0.05 139.8 19.9 4 AdE, Days −30, −2 2.4 ×10⁸ 0.37 0.03 141.7 19.1 5 AdE, −5 Hours 2.4 × 10⁸ 0.38 0.04 146.1 8.7¹There was a stastistically significant difference between group 1 v 3,4, 5; P = 0.41, 0.011 and 0.004, respectively. ²There was astatistically significant difference between groups 1 v 5; P = 0.047.RSV-Tracy Nasal Wash and Lung Lavage Plaque Reduction Titers:

TABLE 9 RSV-Tracy titers in nasal wash fluids on day +4 RSV titer (log₁₀total PFU) in cotton rat Change T test/2 Group Treatment A B C D E FMean SD (log₁₀) v. Gp 1* 1 Buffer Day −2 4.99 5.40 4.98 4.98 5.09 5.025.08 0.16 — — 2 AdE, Day −30 5.04 5.09 4.86 5.07 5.00 4.69 4.96 0.16−0.12 0.230 3 AdE, Day −2 4.86 5.51 5.39 Died 4.94 5.55 5.25 0.32 0.170.280 4 AdE, Days −30, −2 5.45 5.23 4.99 5.13 5.10 5.03 5.15 0.17 0.080.425 5 AdE, −5 Hours 5.56 5.35 5.51 5.45 5.51 5.13 5.42 0.16 0.340.0042 *Minimum detection = 0.7 log10 Total PFU. For statisticalanalysis (Student t test, two tailed) minimum detection (0 plaques) wascounted as 0.35 log10 Total PFU. Additional significant P values: Group5 v 2, 4, P < 0.02.

TABLE 10 RSV-Tracy titers in lung lavage fluids on day +4 RSV titer(log₁₀ PFu/g lung) in cotton rat Change T test/2 Group Treatment A B C DE F Mean SD (log₁₀) v. Gp 1* 1 Buffer Day −2 5.06 5.18 ** 5.06 5.01 5.035.07 0.07 — — 2 AdE, Day −30 4.74 4.59 4.44 4.52 4.65 4.52 4.58 0.11−0.49 0.000010 3 AdE, Day −2 5.03 4.26 4.60 Died 4.60 4.49 4.60 0.28−0.47 0.0063 4 AdE, Days −30, −2 3.95 5.01 4.14 4.54 4.65 4.68 4.49 0.39−0.57 0.0098 5 AdE, −5 Hours 5.02 5.34 4.53 3.88 5.45 4.65 4.81 0.59−0.26 0.357 *Minimum detection 1.3 log10/g lung. **There were no plaquesalthough there was virus in the nasal fluid. Therefore, it was assumedthat the lungs were not infected or technical error. Did not include inthere data for analysis. For Statistical analysis (Student t test,two-tailed) minimal detection (0 plaques) was counted as 1.1 log10/glung. There was no additional significant P values.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

1-19. (canceled)
 20. A method of inducing an immune response againstcoronavirus in a mammalian subject in need thereof, comprising:administering intranasally an effective amount of an E1 and/or E3deleted adenovirus vector that contains and expresses a coronavirusantigen codon optimized for the mammalian subject, wherein the immuneresponse is a protective response that begins within twenty-four hoursof administration.
 21. The method of claim 20, wherein the coronavirusantigen is from SARS-Cov.
 22. The method of claim 20, wherein theeffective amount is at least 10⁷ infectious units (ifu) of E1 and/or E3deleted adenovirus, at least 10⁸ infectious units (ifu) of E1 and/or E3deleted adenovirus or at least 10⁹ infectious units (ifu) of E1 and/orE3 deleted adenovirus.
 23. The method of claim 20, wherein the immuneresponse lasts at least 21 days.
 24. The method of claim 20, wherein thesubject in need thereof is an adult.
 25. The method of claim 20, whereinthe E1 and/or E3 deleted adenovirus vector is administered via at leasttwo steps of administering wherein the administering is 40 days apart,41 days apart, 42 days apart, 43 days apart, 44 days apart, 45 daysapart, 46 days apart, 47 days apart, 48 days apart, 49 days apart or 50days apart.
 26. The method of claim 20, wherein the adenovirus is ahuman adenovirus, a bovine adenovirus, a canine adenovirus, a non-humanprimate adenovirus, a chicken adenovirus, or a porcine or swineadenovirus.
 27. The method of claim 20, wherein the immune response is amucosal immune response.
 28. The method of claim 20, wherein the immuneresponse is non-antigen specific anti-viral immune response.
 29. Themethod of claim 20, wherein the immune response comprises an IgAresponse.
 30. The method of claim 20, wherein the immune responsecomprises a humoral response.
 31. The method of claim 20, wherein theimmune response comprises a cellular response.
 32. A pharmaceuticalformulation configured to induce an immune response against arespiratory pathogen when administered to a mammalian subject in needthereof, comprising: an effective amount of at least 10⁷ infectiousunits (ifu) of E1 and/or E3 deleted adenovirus that contains andexpresses a coronavirus antigen codon optimized for the mammaliansubject, wherein the effective amount induces a protective immuneresponse within 24 hours of administration; and, a pharmaceuticallyacceptable diluent or carrier.
 33. The formulation of claim 32, whereinthe coronavirus antigen is from SARS-Cov.
 34. The formulation of claim32, wherein the effective amount is at least 10⁸ infectious units (ifu)of E1 and/or E3 deleted adenovirus or at least 10⁹ infectious units(ifu) of E1 and/or E3 deleted adenovirus.
 35. The formulation of claim32, wherein the adenovirus is a human adenovirus, a bovine adenovirus, acanine adenovirus, a non-human primate adenovirus, a chicken adenovirus,or a porcine or swine adenovirus.
 36. The formulation of claim 32,wherein the immune response is a mucosal immune response, optionallycomprising an IgA response.
 37. The formulation of claim 32, wherein theimmune response is non-antigen specific anti-viral immune response. 38.The formulation of claim 32, wherein the immune response comprises ahumoral response.
 39. The formulation of claim 32, wherein the immuneresponse comprises a cellular response.